PUFA polyketide synthase systems and uses thereof

ABSTRACT

The invention generally relates to polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systems isolated from or derived from non-bacterial organisms, to homologues thereof, to isolated nucleic acid molecules and recombinant nucleic acid molecules encoding biologically active domains of such a PUFA PKS system, to genetically modified organisms comprising PUFA PKS systems, to methods of making and using such systems for the production of bioactive molecules of interest, and to novel methods for identifying new bacterial and non-bacterial microorganisms having such a PUFA PKS system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/124,800, filed Apr. 16, 2002, now U.S. Pat. No. 7,247,461, entitled“PUFA Polyketide Synthase Systems and Uses Thereof,” which claims thebenefit of priority under 35 U.S.C. § 119(e) to: U.S. ProvisionalApplication Ser. No. 60/284,066, filed Apr. 16, 2001, entitled “APolyketide Synthase System and Uses Thereof”; U.S. ProvisionalApplication Ser. No. 60/298,796, filed Jun. 15, 2001, entitled “APolyketide Synthase System and Uses Thereof”; and U.S. ProvisionalApplication Ser. No. 60/323,269, filed Sep. 18, 2001, entitled“Thraustochytrium PUFA PKS System and Uses Thereof”. U.S. applicationSer. No. 10/124,800, is also a continuation-in-part of U.S. applicationSer. No. 09/231,899, now U.S. Pat. No. 6,566,583, filed Jan. 14, 1999,entitled “Schizochytrium PKS Genes”. Each of the above-identified patentapplications is incorporated herein by reference in its entirety.

This application does not claim the benefit of priority from U.S.application Ser. No. 09/090,793, filed Jun. 4, 1998, now U.S. Pat. No.6,140,486, although U.S. application Ser. No. 09/090,793 is incorporatedherein by reference in its entirety.

REFERENCE TO SEQUENCE LISITNG

This application contains a Sequence Listing submitted as an electronictext file named “2997-29_corrected_ST25.txt”, having a size in bytes of280kb, and created on 4 Mar. 2007. The information contained in thiselectronic file is hereby incorporated by reference in its entiretypursuant to 37 CFR §1.52(e)(5).

FIELD OF THE INVENTION

This invention relates to polyunsaturated fatty acid (PUFA) polyketidesynthase (PKS) systems from microorganisms, including eukaryoticorganisms, such as Thraustochytrid microorganisms. More particularly,this invention relates to nucleic acids encoding non-bacterial PUFA PKSsystems, to non-bacterial PUFA PKS systems, to genetically modifiedorganisms comprising non-bacterial PUFA PKS systems, and to methods ofmaking and using the non-bacterial PUFA PKS systems disclosed herein.This invention also relates to a method to identify bacterial andnon-bacterial microorganisms comprising PUFA PKS systems.

BACKGROUND OF THE INVENTION

Polyketide synthase (PKS) systems are generally known in the art asenzyme complexes derived from fatty acid synthase (FAS) systems, butwhich are often highly modified to produce specialized products thattypically show little resemblance to fatty acids. Researchers haveattempted to exploit polyketide synthase (PKS) systems that have beendescribed in the literature as falling into one of three basic types,typically referred to as: Type II, Type I and modular. The Type IIsystem is characterized by separable proteins, each of which carries outa distinct enzymatic reaction. The enzymes work in concert to producethe end product and each individual enzyme of the system typicallyparticipates several times in the production of the end product. Thistype of system operates in a manner analogous to the fatty acid synthase(FAS) systems found in plants and bacteria. Type I PKS systems aresimilar to the Type II system in that the enzymes are used in aniterative fashion to produce the end product. The Type I differs fromType II in that enzymatic activities, instead of being associated withseparable proteins, occur as domains of larger proteins. This system isanalogous to the Type I FAS systems found in animals and fungi.

In contrast to the Type I and II systems, in modular PKS systems, eachenzyme domain is used only once in the production of the end product.The domains are found in very large proteins and the product of eachreaction is passed on to another domain in the PKS protein.Additionally, in all of the PKS systems described above, if acarbon-carbon double bond is incorporated into the end product, it isalways in the trans configuration.

In the Type I and Type II PKS systems described above, the same set ofreactions is carried out in each cycle until the end product isobtained. There is no allowance for the introduction of unique reactionsduring the biosynthetic procedure. The modular PKS systems require hugeproteins that do not utilize the economy of iterative reactions (i.e., adistinct domain is required for each reaction). Additionally, as statedabove, carbon-carbon double bonds are introduced in the transconfiguration in all of the previously described PKS systems.

Polyunsaturated fatty acids (PUFAs) are critical components of membranelipids in most eukaryotes (Lauritzen et al., Prog. Lipid Res. 40 1(2001); McConn et al., Plant J. 15, 521 (1998)) and are precursors ofcertain hormones and signaling molecules (Heller et al., Drugs 55, 487(1998); Creelman et al., Annu. Rev. Plant Physiol. Plant Mol. Biol. 48,355 (1997)). Known pathways of PUFA synthesis involve the processing ofsaturated 16:0 or 18:0 fatty acids (the abbreviation X:Y indicates anacyl group containing X carbon atoms and Y cis double bonds; double-bondpositions of PUFAs are indicated relative to the methyl carbon of thefatty acid chain (ω3 or ω6) with systematic methylene interruption ofthe double bonds) derived from fatty acid synthase (FAS) by elongationand aerobic desaturation reactions (Sprecher, Curr. Opin. Clin. Nutr.Metab. Care 2, 135 (1999); Parker-Barnes et al., Proc. Natl. Acad. Sci.USA 97, 8284 (2000); Shanklin et al., Annu. Rev. Plant Physiol. PlantNol. Biol. 49, 611 (1998)). Starting from acetyl-CoA, the synthesis ofDHA requires approximately 30 distinct enzyme activities and nearly 70reactions including the four repetitive steps of the fatty acidsynthesis cycle. Polyketide synthases (PKSs) carry out some of the samereactions as FAS (Hopwood et al., Annu. Rev. Genet. 24, 37 (1990);Bentley et al., Annu. Rev. Microbiol. 53, 411 (1999)) and use the samesmall protein (or domain), acyl carrier protein (ACP), as a covalentattachment site for the growing carbon chain. However, in these enzymesystems, the complete cycle of reduction, dehydration and reduction seenin FAS is often abbreviated so that a highly derivatized carbon chain isproduced, typically containing many keto- and hydroxy-groups as well ascarbon-carbon double bonds in the trans configuration. The linearproducts of PKSs are often cyclized to form complex biochemicals thatinclude antibiotics and many other secondary products (Hopwood et al.,(1990) supra; Bentley et al., (1999), supra; Keating et al., Curr. Opin.Chem. Biol. 3, 598 (1999)).

Very long chain PUFAs such as docosahexaenoic acid (DHA; 22:6ω3) andeicosapentaenoic acid (EPA; 20:5ω3) have been reported from severalspecies of marine bacteria, including Shewanella sp (Nichols et al.,Curr. Op. Biotechnol. 10, 240 (1999); Yazawa, Lipids 31, S (1996);DeLong et al., Appl. Environ. Microbiol. 51, 730 (1986)). Analysis of agenomic fragment (cloned as plasmid pEPA) from Shewanella sp. strainSCRC2738 led to the identification of five open reading frames (Orfs),totaling 20 Kb, that are necessary and sufficient for EPA production inE. coli (Yazawa, (1996), supra). Several of the predicted proteindomains were homologues of FAS enzymes, while other regions showed nohomology to proteins of known function. On the basis of theseobservations and biochemical studies, it was suggested that PUFAsynthesis in Shewanella involved the elongation of 16- or 18-carbonfatty acids produced by FAS and the insertion of double bonds byundefined aerobic desaturases (Watanabe et al., J. Biochem. 122, 467(1997)). The recognition that this hypothesis was incorrect began with areexamination of the protein sequences encoded by the five ShewanellaOrfs. At least 11 regions within the five Orfs were identifiable asputative enzyme domains (See Metz et al., Science 293:290-293 (2001)).When compared with sequences in the gene databases, seven of these weremore strongly related to PKS proteins than to FAS proteins. Included inthis group were domains putatively encoding malonyl-CoA:ACPacyltransferase (MAT), 3-ketoacyl-ACP synthase (KS), 3-ketoacyl-ACPreductase (KR), acyltransferase (AT), phosphopantetheine transferase,chain length (or chain initiation) factor (CLF) and a highly unusualcluster of six ACP domains (i.e., the presence of more than twoclustered ACP domains has not previously been reported in PKS or FASsequences). However, three regions were more highly homologous tobacterial FAS proteins. One of these was similar to the newly-describedTriclosan-resistant enoyl reductase (ER) from Streptococcus pneumoniae(Heath et al., Nature 406, 145 (2000)); comparison of ORF8 peptide withthe S. pneumoniae enoyl reductase using the LALIGN program (matrix,BLOSUM50; gap opening penalty, −10; elongation penalty −1) indicated 49%similarity over a 386aa overlap). Two regions were homologues of the E.coli FAS protein encoded by fabA, which catalyzes the synthesis oftrans-2-decenoyl-ACP and the reversible isomerization of this product tocis-3-decenoyl-ACP (Heath et al., J. Biol. Chem., 271, 27795 (1996)). Onthis basis, it seemed likely that at least some of the double bonds inEPA from Shewanella are introduced by a dehydrase-isomerase mechanismcatalyzed by the FabA-like domains in Orf7.

Anaerobically-grown E. coli cells harboring the pEPA plasmid accumulatedEPA to the same levels as aerobic cultures (Metz et al., 2001, supra),indicating that an oxygen-dependent desaturase is not involved in EPAsynthesis. When pEPA was introduced into a fabB⁻ mutant of E. coli,which is unable to synthesize monounsaturated fatty acids and requiresunsaturated fatty acids for growth, the resulting cells lost their fattyacid auxotrophy. They also accumulated much higher levels of EPA thanother pEPA-containing strains, suggesting that EPA competes withendogenously produced monounsaturated fatty acids for transfer toglycerolipids. When pEPA-containing E. coli cells were grown in thepresence of [¹³C]-acetate, the data from ¹³C-NMR analysis of purifiedEPA from the cells confirmed the identity of EPA and provided evidencethat this fatty acid was synthesized from acetyl-CoA and malonyl-CoA(See Metz et al., 2001, supra). A cell-free homogenate frompEPA-containing fabB⁻ cells synthesized both EPA and saturated fattyacids from [¹⁴C]-malonyl-CoA. When the homogenate was separated into a200,000×g high-speed pellet and a membrane-free supernatant fraction,saturated fatty acid synthesis was confined to the supernatant,consistent with the soluble nature of the Type II FAS enzymes (Magnusonet al., Microbiol. Rev. 57, 522 (1993)). Synthesis of EPA was found onlyin the high-speed pellet fraction, indicating that EPA synthesis canoccur without reliance on enzymes of the E. coli FAS or on solubleintermediates (such as 16:0-ACP) from the cytoplasmic fraction. Sincethe proteins encoded by the Shewanella EPA genes are not particularlyhydrophobic, restriction of EPA synthesis activity to this fraction mayreflect a requirement for a membrane-associated acyl acceptor molecule.Additionally, in contrast to the E. coli FAS, EPA synthesis isspecifically NADPH-dependent and does not require NADH. All theseresults are consistent with the pEPA genes encoding a multifunctionalPKS that acts independently of FAS, elongase, and desaturase activitiesto synthesize EPA directly. It is likely that the PKS pathway for PUFAsynthesis that has been identified in Shewanella is widespread in marinebacteria. Genes with high homology to the Shewanella gene cluster havebeen identified in Photobacterium profundum (Allen et al., Appli.Environ. Microbiol. 65:1710 (1999)) and in Moritella marina (Vibriomarinus) (Tanaka et al., Biotechnol. Lett. 21:939 (1999)).

The biochemical and molecular-genetic analyses performed with Shewanellaprovide compelling evidence for polyketide synthases that are capable ofsynthesizing PUFAs from malonyl-CoA. A complete scheme for synthesis ofEPA by the Shewanella PKS has been proposed. The identification ofprotein domains homologous to the E. coli FabA protein, and theobservation that bacterial EPA synthesis occurs anaerobically, provideevidence for one mechanism wherein the insertion of cis double bondsoccurs through the action of a bifunctional dehydratase/2-trans, 3-cisisomerase (DH/2,3I). In E. coli, condensation of the 3-cis acylintermediate with malonyl-ACP requires a particular ketoacyl-ACPsynthase and this may provide a rationale for the presence of two KS inthe Shewanella gene cluster (in Orf 5 and Orf 7). However, the PKS cycleextends the chain in two-carbon increments while the double bonds in theEPA product occur at every third carbon. This disjunction can be solvedif the double bonds at C-14 and C-8 of EPA are generated by 2-trans,2-cis isomerization (DH/2,2I) followed by incorporation of the cisdouble bond into the elongating fatty acid chain. The enzymaticconversion of a trans double bond to the cis configuration without bondmigration is known to occur, for example, in the synthesis of11-cis-retinal in the retinoid cycle (Jang et al., J. Biol. Chem. 275,28128 (2000)). Although such an enzyme function has not yet beenidentified in the Shewanella PKS, it may reside in one of the unassignedprotein domains.

The PKS pathways for PUFA synthesis in Shewanella and another marinebacteria, Vibrio marinus, are described in detail in U.S. Pat. No.6,140,486 (issued from U.S. application Ser. No. 09/090,793, filed Jun.4, 1998, entitled “Production of Polyunsaturated Fatty Acids byExpression of Polyketide-like Synthesis Genes in Plants”, which isincorporated herein by reference in its entirety).

Polyunsaturated fatty acids (PUFAs) are considered to be useful fornutritional, pharmaceutical, industrial, and other purposes. Anexpansive supply of PUFAs from natural sources and from chemicalsynthesis are not sufficient for commercial needs. Because a number ofseparate desaturase and elongase enzymes are required for fatty acidsynthesis from linoleic acid (LA, 18:2 Δ 9, 12), common in most plantspecies, to the more saturated and longer chain PUFAs, engineering planthost cells for the expression of PUFAs such as EPA and DHA may requireexpression of five or six separate enzyme activities to achieveexpression, at least for EPA and DHA. Additionally, for production ofuseable quantities of such PUFAs, additional engineering efforts may berequired, for instance the down regulation of enzymes competing forsubstrate, engineering of higher enzyme activities such as bymutagenesis or targeting of enzymes to plastid organelles. Therefore itis of interest to obtain genetic material involved in PUFA biosynthesisfrom species that naturally produce these fatty acids and to express theisolated material alone or in combination in a heterologous system whichcan be manipulated to allow production of commercial quantities ofPUFAs.

The discovery of a PUFA PKS system in marine bacteria such as Shewanellaand Vibrio marinus (see U.S. Pat. No. 6,140,486, ibid.) provides aresource for new methods of commercial PUFA production. However, thesemarine bacteria have limitations which will ultimately restrict theirusefulness on a commercial level. First, although U.S. Pat. No.6,140,486 discloses that the marine bacteria PUFA PKS systems can beused to genetically modify plants, the marine bacteria naturally liveand grow in cold marine environments and the enzyme systems of thesebacteria do not function well above 30° C. In contrast, many cropplants, which are attractive targets for genetic manipulation using thePUFA PKS system, have normal growth conditions at temperatures above 30°C. and ranging to higher than 40° C. Therefore, the marine bacteria PUFAPKS system is not predicted to be readily adaptable to plant expressionunder normal growth conditions. Moreover, the marine bacteria PUFA PKSgenes, being from a bacterial source, may not be compatible with thegenomes of eukaryotic host cells, or at least may require significantadaptation to work in eukaryotic hosts. Additionally, the known marinebacteria PUFA PKS systems do not directly produce triglycerides, whereasdirect production of triglycerides would be desirable becausetriglycerides are a lipid storage product in microorganisms and as aresult can be accumulated at very high levels (e.g. up to 80-85% of cellweight) in microbial/plant cells (as opposed to a “structural” lipidproduct (e.g. phospholipids) which can generally only accumulate at lowlevels (e.g. less than 10-15% of cell weight at maximum)).

Therefore, there is a need in the art for other PUFA PKS systems havinggreater flexibility for commercial use.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to an isolated nucleicacid molecule comprising a nucleic acid sequence chosen from: (a) anucleic acid sequence encoding an amino acid sequence selected from thegroup consisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, andbiologically active fragments thereof; (b) a nucleic acid sequenceencoding an amino acid sequence selected from the group consisting of:SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:20, SEQID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, and biologically active fragments thereof; (c) a nucleic acidsequence encoding an amino acid sequence that is at least about 60%identical to at least 500 consecutive amino acids of the amino acidsequence of (a), wherein the amino acid sequence has a biologicalactivity of at least one domain of a polyunsaturated fatty acid (PUFA)polyketide synthase (PKS) system; (d) a nucleic acid sequence encodingan amino acid sequence that is at least about 60% identical to the aminoacid sequence of (b), wherein the amino acid sequence has a biologicalactivity of at least one domain of a polyunsaturated fatty acid (PUFA)polyketide synthase (PKS) system; and (e) a nucleic acid sequence thatis fully complementary to the nucleic acid sequence of (a), (b), (c), or(d). In alternate aspects, the nucleic acid sequence encodes an aminoacid sequence that is at least about 70% identical, or at least about80% identical, or at least about 90% identical, or is identical to: (1)at least 500 consecutive amino acids of an amino acid sequence selectedfrom the group consisting of: SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6;and/or (2) a nucleic acid sequence encoding an amino acid sequence thatis at least about 70% identical to an amino acid sequence selected fromthe group consisting of: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, and SEQ ID NO:32. In a preferred embodiment, thenucleic acid sequence encodes an amino acid sequence chosen from: SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13,SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26,SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32 and/or biologically activefragments thereof. In one aspect, the nucleic acid sequence is chosenfrom: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ ID NO:31.

Another embodiment of the present invention relates to a recombinantnucleic acid molecule comprising the nucleic acid molecule as describedabove, operatively linked to at least one transcription controlsequence. In another embodiment, the present invention relates to arecombinant cell transfected with the recombinant nucleic acid moleculedescribed directly above.

Yet another embodiment of the present invention relates to a geneticallymodified microorganism, wherein the microorganism expresses a PKS systemcomprising at least one biologically active domain of a polyunsaturatedfatty acid (PUFA) polyketide synthase (PKS) system. The at least onedomain of the PUFA PKS system is encoded by a nucleic acid sequencechosen from: (a) a nucleic acid sequence encoding at least one domain ofa polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systemfrom a Thraustochytrid microorganism; (b) a nucleic acid sequenceencoding at least one domain of a PUFA PKS system from a microorganismidentified by the screening method of the present invention; (c) anucleic acid sequence encoding an amino acid sequence selected from thegroup consisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, andbiologically active fragments thereof, (d) a nucleic acid sequenceencoding an amino acid sequence selected from the group consisting of:SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:20, SEQID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, and biologically active fragments thereof; (e) a nucleic acidsequence encoding an amino acid sequence that is at least about 60%identical to at least 500 consecutive amino acids of an amino acidsequence selected from the group consisting of: SEQ ID NO:2, SEQ IDNO:4, and SEQ ID NO:6; wherein the amino acid sequence has a biologicalactivity of at least one domain of a PUFA PKS system; and, (f) a nucleicacid sequence encoding an amino acid sequence that is at least about 60%identical to an amino acid sequence selected from the group consistingof: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30,and SEQ ID NO:32; wherein the amino acid sequence has a biologicalactivity of at least one domain of a PUFA PKS system. In thisembodiment, the microorganism is genetically modified to affect theactivity of the PKS system. The screening method of the presentinvention referenced in (b) above comprises: (i) selecting amicroorganism that produces at least one PUFA; and, (ii) identifying amicroorganism from (i) that has an ability to produce increased PUFAsunder dissolved oxygen conditions of less than about 5% of saturation inthe fermentation medium, as compared to production of PUFAs by themicroorganism under dissolved oxygen conditions of greater than 5% ofsaturation, and more preferably 10% of saturation, and more preferablygreater than 15% of saturation, and more preferably greater than 20% ofsaturation in the fermentation medium.

In one aspect, the microorganism endogenously expresses a PKS systemcomprising the at least one domain of the PUFA PKS system, and whereinthe genetic modification is in a nucleic acid sequence encoding the atleast one domain of the PUFA PKS system. For example, the geneticmodification can be in a nucleic acid sequence that encodes a domainhaving a biological activity of at least one of the following proteins:malonyl-CoA:ACP acyltransferase (MAT), β-keto acyl-ACP synthase (KS),ketoreductase (KR), acyltransferase (AT), FabA-like β-hydroxy acyl-ACPdehydrase (DH), phosphopantetheine transferase, chain length factor(CLF), acyl carrier protein (ACP), enoyl ACP-reductase (ER), an enzymethat catalyzes the synthesis of trans-2-decenoyl-ACP, an enzyme thatcatalyzes the reversible isomerization of trans-2-decenoyl-ACP tocis-3-decenoyl-ACP, and an enzyme that catalyzes the elongation ofcis-3-decenoyl-ACP to cis-vaccenic acid. In one aspect, the geneticmodification is in a nucleic acid sequence that encodes an amino acidsequence selected from the group consisting of: (a) an amino acidsequence that is at least about 70% identical, and preferably at leastabout 80% identical, and more preferably at least about 90% identicaland more preferably identical to at least 500 consecutive amino acids ofan amino acid sequence selected from the group consisting of: SEQ IDNO:2, SEQ ID NO:4, and SEQ ID NO:6; wherein the amino acid sequence hasa biological activity of at least one domain of a PUFA PKS system; and,(b) an amino acid sequence that is at least about 70% identical, andpreferably at least about 80% identical, and more preferably at leastabout 90% identical and more preferably identical to an amino acidsequence selected from the group consisting of: SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:32;wherein the amino acid sequence has a biological activity of at leastone domain of a PUFA PKS system.

In one aspect, the genetically modified microorganism is aThraustochytrid, which can include, but is not limited to, aThraustochytrid from a genus chosen from Schizochytrium andThraustochytrium. In another aspect, the microorganism has been furthergenetically modified to recombinantly express at least one nucleic acidmolecule encoding at least one biologically active domain from abacterial PUFA PKS system, from a Type I PKS system, from a Type II PKSsystem, and/or from a modular PKS system.

In another aspect of this embodiment, the microorganism endogenouslyexpresses a PUFA PKS system comprising the at least one biologicallyactive domain of a PUFA PKS system, and wherein the genetic modificationcomprises expression of a recombinant nucleic acid molecule selectedfrom the group consisting of a recombinant nucleic acid moleculeencoding at least one biologically active domain from a second PKSsystem and a recombinant nucleic acid molecule encoding a protein thataffects the activity of the PUFA PKS system. Preferably, the recombinantnucleic acid molecule comprises any one of the nucleic acid sequencesdescribed above.

In one aspect of this embodiment, the recombinant nucleic acid moleculeencodes a phosphopantetheine transferase. In another aspect, therecombinant nucleic acid molecule comprises a nucleic acid sequenceencoding at least one biologically active domain from a bacterial PUFAPKS system, from a type I PKS system, from a type II PKS system, and/orfrom a modular PKS system.

In another aspect of this embodiment, the microorganism is geneticallymodified by transfection with a recombinant nucleic acid moleculeencoding the at least one domain of a polyunsaturated fatty acid (PUFA)polyketide synthase (PKS) system. Such a recombinant nucleic acidmolecule can include any recombinant nucleic acid molecule comprisingany of the nucleic acid sequences described above. In one aspect, themicroorganism has been further genetically modified to recombinantlyexpress at least one nucleic acid molecule encoding at least onebiologically active domain from a bacterial PUFA PKS system, from a TypeI PKS system, from a Type II PKS system, or from a modular PKS system.

Yet another embodiment of the present invention relates to a geneticallymodified plant, wherein the plant has been genetically modified torecombinantly express a PKS system comprising at least one biologicallyactive domain of a polyunsaturated fatty acid (PUFA) polyketide synthase(PKS) system. The domain can be encoded by any of the nucleic acidsequences described above. In one aspect, the plant has been furthergenetically modified to recombinantly express at least one nucleic acidmolecule encoding at least one biologically active domain from abacterial PUFA PKS system, from a Type I PKS system, from a Type II PKSsystem, and/from a modular PKS system.

Another embodiment of the present invention relates to a method toidentify a microorganism that has a polyunsaturated fatty acid (PUFA)polyketide synthase (PKS) system. The method includes the steps of: (a)selecting a microorganism that produces at least one PUFA; and, (b)identifying a microorganism from (a) that has an ability to produceincreased PUFAs under dissolved oxygen conditions of less than about 5%of saturation in the fermentation medium, as compared to production ofPUFAs by the microorganism under dissolved oxygen conditions of greaterthan 5% of saturation, more preferably 10% of saturation, morepreferably greater than 15% of saturation and more preferably greaterthan 20% of saturation in the fermentation medium. A microorganism thatproduces at least one PUFA and has an ability to produce increased PUFAsunder dissolved oxygen conditions of less than about 5% of saturation isidentified as a candidate for containing a PUFA PKS system.

In one aspect of this embodiment, step (b) comprises identifying amicroorganism from (a) that has an ability to produce increased PUFAsunder dissolved oxygen conditions of less than about 2% of saturation,and more preferably under dissolved oxygen conditions of less than about1% of saturation, and even more preferably under dissolved conditions ofabout 0% of saturation.

In another aspect of this embodiment, the microorganism selected in (a)has an ability to consume bacteria by phagocytosis. In another aspect,the microorganism selected in (a) has a simple fatty acid profile. Inanother aspect, the microorganism selected in (a) is a non-bacterialmicroorganism. In another aspect, the microorganism selected in (a) is aeukaryote.

In another aspect, the microorganism selected in (a) is a member of theorder Thraustochytriales. In another aspect, the microorganism selectedin (a) has an ability to produce PUFAs at a temperature greater thanabout 15° C., and preferably greater than about 20° C., and morepreferably greater than about 25° C., and even more preferably greaterthan about 30° C. In another aspect, the microorganism selected in (a)has an ability to produce bioactive compounds (e.g., lipids) of interestat greater than 5% of the dry weight of the organism, and morepreferably greater than 10% of the dry weight of the organism. In yetanother aspect, the microorganism selected in (a) contains greater than30% of its total fatty acids as C14:0, C16:0 and C16:1 while alsoproducing at least one long chain fatty acid with three or moreunsaturated bonds, and preferably, the microorganism selected in (a)contains greater than 40% of its total fatty acids as C14:0, C16:0 andC16:1 while also producing at least one long chain fatty acid with threeor more unsaturated bonds. In another aspect, the microorganism selectedin (a) contains greater than 30% of its total fatty acids as C14:0,C16:0 and C16:1 while also producing at least one long chain fatty acidwith four or more unsaturated bonds, and more preferably while alsoproducing at least one long chain fatty acid with five or moreunsaturated bonds.

In another aspect of this embodiment, the method further comprises step(c) of detecting whether the organism comprises a PUFA PKS system. Inthis aspect, the step of detecting can include detecting a nucleic acidsequence in the microorganism that hybridizes under stringent conditionswith a nucleic acid sequence encoding an amino acid sequence from aThraustochytrid PUFA PKS system. Alternatively, the step of detectingcan include detecting a nucleic acid sequence in the organism that isamplified by oligonucleotide primers from a nucleic acid sequence from aThaustochytrid PUFA PKS system.

Another embodiment of the present invention relates to a microorganismidentified by the screening method described above, wherein themicroorganism is genetically modified to regulate the production ofmolecules by the PUFA PKS system.

Yet another embodiment of the present invention relates to a method toproduce a bioactive molecule that is produced by a polyketide synthasesystem. The method includes the step of culturing under conditionseffective to produce the bioactive molecule a genetically modifiedorganism that expresses a PKS system comprising at least onebiologically active domain of a polyunsaturated fatty acid (PUFA)polyketide synthase (PKS) system. The domain of the PUFA PKS system isencoded by any of the nucleic acid sequences described above.

In one aspect of this embodiment, the organism endogenously expresses aPKS system comprising the at least one domain of the PUFA PKS system,and the genetic modification is in a nucleic acid sequence encoding theat least one domain of the PUFA PKS system. For example, the geneticmodification can change at least one product produced by the endogenousPKS system, as compared to a wild-type organism.

In another aspect of this embodiment, the organism endogenouslyexpresses a PKS system comprising the at least one biologically activedomain of the PUFA PKS system, and the genetic modification comprisestransfection of the organism with a recombinant nucleic acid moleculeselected from the group consisting of: a recombinant nucleic acidmolecule encoding at least one biologically active domain from a secondPKS system and a recombinant nucleic acid molecule encoding a proteinthat affects the activity of the PUFA PKS system. For example, thegenetic modification can change at least one product produced by theendogenous PKS system, as compared to a wild-type organism.

In yet another aspect of this embodiment, the organism is geneticallymodified by transfection with a recombinant nucleic acid moleculeencoding the at least one domain of the polyunsaturated fatty acid(PUFA) polyketide synthase (PKS) system. In another aspect, the organismproduces a polyunsaturated fatty acid (PUFA) profile that differs fromthe naturally occurring organism without a genetic modification. Inanother aspect, the organism endogenously expresses a non-bacterial PUFAPKS system, and wherein the genetic modification comprises substitutionof a domain from a different PKS system for a nucleic acid sequenceencoding at least one domain of the non-bacterial PUFA PKS system.

In yet another aspect, the organism endogenously expresses anon-bacterial PUFA PKS system that has been modified by transfecting theorganism with a recombinant nucleic acid molecule encoding a proteinthat regulates the chain length of fatty acids produced by the PUFA PKSsystem. For example, the recombinant nucleic acid molecule encoding aprotein that regulates the chain length of fatty acids can replace anucleic acid sequence encoding a chain length factor in thenon-bacterial PUFA PKS system. In another aspect, the protein thatregulates the chain length of fatty acids produced by the PUFA PKSsystem is a chain length factor. In another aspect, the protein thatregulates the chain length of fatty acids produced by the PUFA PKSsystem is a chain length factor that directs the synthesis of C20 units.

In one aspect, the organism expresses a non-bacterial PUFA PKS systemcomprising a genetic modification in a domain chosen from: a domainencoding FabA-like β-hydroxy acyl-ACP dehydrase (DH) domain and a domainencoding β-ketoacyl-ACP synthase (KS), wherein the modification altersthe ratio of long chain fatty acids produced by the PUFA PKS system ascompared to in the absence of the modification. In one aspect, themodification comprises substituting a DH domain that does not possessisomerization activity for a FabA-like β-hydroxy acyl-ACP dehydrase (DH)in the non-bacterial PUFA PKS system. In another aspect, themodification is selected from the group consisting of a deletion of allor a part of the domain, a substitution of a homologous domain from adifferent organism for the domain, and a mutation of the domain.

In another aspect, the organism expresses a PKS system and the geneticmodification comprises substituting a FabA-like β-hydroxy acyl-ACPdehydrase (DH) domain from a PUFA PKS system for a DH domain that doesnot posses isomerization activity.

In another aspect, the organism expresses a non-bacterial PUFA PKSsystem comprising a modification in an enoyl-ACP reductase (ER) domain,wherein the modification results in the production of a differentcompound as compared to in the absence of the modification. For example,the modification can be selected from the group consisting of a deletionof all or a part of the ER domain, a substitution of an ER domain from adifferent organism for the ER domain, and a mutation of the ER domain.

In one aspect, the bioactive molecule produced by the present method caninclude, but is not limited to, an anti-inflammatory formulation, achemotherapeutic agent, an active excipient, an osteoporosis drug, ananti-depressant, an anti-convulsant, an anti-Heliobactor pylori drug, adrug for treatment of neurodegenerative disease, a drug for treatment ofdegenerative liver disease, an antibiotic, and a cholesterol loweringformulation. In one aspect, the bioactive molecule is a polyunsaturatedfatty acid (PUFA). In another aspect, the bioactive molecule is amolecule including carbon-carbon double bonds in the cis configuration.In another aspect, the bioactive molecule is a molecule including adouble bond at every third carbon.

In one aspect of this embodiment, the organism is a microorganism, andin another aspect, the organism is a plant.

Another embodiment of the present invention relates to a method toproduce a plant that has a polyunsaturated fatty acid (PUFA) profilethat differs from the naturally occurring plant, comprising geneticallymodifying cells of the plant to express a PKS system comprising at leastone recombinant nucleic acid molecule comprising a nucleic acid sequenceencoding at least one biologically active domain of a PUFA PKS system.The domain of the PUFA PKS system is encoded by any of the nucleic acidsequences described above.

Yet another embodiment of the present invention relates to a method tomodify an endproduct containing at least one fatty acid, comprisingadding to the endproduct an oil produced by a recombinant host cell thatexpresses at least one recombinant nucleic acid molecule comprising anucleic acid sequence encoding at least one biologically active domainof a PUFA PKS system. The domain of a PUFA PKS system is encoded by anyof the nucleic acid sequences described above. In one aspect, theendproduct is selected from the group consisting of a dietarysupplement, a food product, a pharmaceutical formulation, a humanizedanimal milk, and an infant formula. A pharmaceutical formulation caninclude, but is not limited to: an anti-inflammatory formulation, achemotherapeutic agent, an active excipient, an osteoporosis drug, ananti-depressant, an anti-convulsant, an anti-Heliobactor pylori drug, adrug for treatment of neurodegenerative disease, a drug for treatment ofdegenerative liver disease, an antibiotic, and a cholesterol loweringformulation. In one aspect, the endproduct is used to treat a conditionselected from the group consisting of: chronic inflammation, acuteinflammation, gastrointestinal disorder, cancer, cachexia, cardiacrestenosis, neurodegenerative disorder, degenerative disorder of theliver, blood lipid disorder, osteoporosis, osteoarthritis, autoimmunedisease, preeclampsia, preterm birth, age related maculopathy, pulmonarydisorder, and peroxisomal disorder.

Yet another embodiment of the present invention relates to a method toproduce a humanized animal milk, comprising genetically modifyingmilk-producing cells of a milk-producing animal with at least onerecombinant nucleic acid molecule comprising a nucleic acid sequenceencoding at least one biologically active domain of a PUFA PKS system.The domain of the PUFA PKS system is encoded by any of the nucleic acidsequences described above.

Yet another embodiment of the present invention relates to a methodproduce a recombinant microbe, comprising genetically modifyingmicrobial cells to express at least one recombinant nucleic acidmolecule comprising a comprising a nucleic acid sequence encoding atleast one biologically active domain of a PUFA PKS system. The domain ofthe PUFA PKS system is encoded by any of the nucleic acid sequencesdescribed above.

Yet another embodiment of the present invention relates to a recombinanthost cell which has been modified to express a polyunsaturated fattyacid (PUFA) polyketide synthase (PKS) system, wherein the PKS catalyzesboth iterative and non-iterative enzymatic reactions. The PUFA PKSsystem comprises: (a) at least two enoyl ACP-reductase (ER) domains; (b)at least six acyl carrier protein (ACP) domains; (c) at least two β-ketoacyl-ACP synthase (KS) domains; (d) at least one acyltransferase (AT)domain; (e) at least one ketoreductase (KR) domain; (f) at least twoFabA-like β-hydroxy acyl-ACP dehydrase (DH) domains; (g) at least onechain length factor (CLF) domain; and (h) at least one malonyl-CoA:ACPacyltransferase (MAT) domain. In one aspect, the PUFA PKS system is aeukaryotic PUFA PKS system. In another aspect, the PUFA PKS system is analgal PUFA PKS system, and preferably a Thraustochytriales PUFA PKSsystem, which can include, but is not limited to, a Schizochytrium PUFAPKS system or a Thraustochytrium PUFA PKS system.

In this embodiment, the PUFA PKS system can be expressed in aprokaryotic host cell or in a eukaryotic host cell. In one aspect, thehost cell is a plant cell. Accordingly, one embodiment of the inventionis a method to produce a product containing at least one PUFA,comprising growing a plant comprising such a plant cell under conditionseffective to produce the product. The host cell is a microbial cell andin this case, one embodiment of the present invention is a method toproduce a product containing at least one PUFA, comprising culturing aculture containing such a microbial cell under conditions effective toproduce the product. In one aspect, the PKS system catalyzes the directproduction of triglycerides.

Yet another embodiment of the present invention relates to a geneticallymodified microorganism comprising a polyunsaturated fatty acid (PUFA)polyketide synthase (PKS) system, wherein the PKS catalyzes bothiterative and non-iterative enzymatic reactions. The PUFA PKS systemcomprises: (a) at least two enoyl ACP-reductase (ER) domains; (b) atleast six acyl carrier protein (ACP) domains; (c) at least two β-ketoacyl-ACP synthase (KS) domains; (d) at least one acyltransferase (AT)domain; (e) at least one ketoreductase (KR) domain; (f) at least twoFabA-like β-hydroxy acyl-ACP dehydrase (DH) domains; (g) at least onechain length factor (CLF) domain; and (h) at least one malonyl-CoA:ACPacyltransferase (MAT) domain. The genetic modification affects theactivity of the PUFA PKS system. In one aspect of this embodiment, themicroorganism is a eukaryotic microorganism.

Yet another embodiment of the present invention relates to a recombinanthost cell which has been modified to express a non-bacterialpolyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system,wherein the non-bacterial PUFA PKS catalyzes both iterative andnon-iterative enzymatic reactions. The non-bacterial PUFA PKS systemcomprises: (a) at least one enoyl ACP-reductase (ER) domain; (b)multiple acyl carrier protein (ACP) domains; (c) at least two β-ketoacyl-ACP synthase (KS) domains; (d) at least one acyltransferase (AT)domain; (e) at least one ketoreductase (KR) domain; (f) at least twoFabA-like β-hydroxy acyl-ACP dehydrase (DH) domains; (g) at least onechain length factor (CLF) domain; and (h) at least one malonyl-CoA:ACPacyltransferase (MAT) domain.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical representation of the domain structure of theSchizochytrium PUFA PKS system.

FIG. 2 shows a comparison of PKS domains from Schizochytrium andShewanella.

FIG. 3 shows a comparison of PKS domains from Schizochytrium and arelated PKS system from Nostoc whose product is a long chain fatty acidthat does not contain any double bonds.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to non-bacterial derivedpolyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systems, togenetically modified organisms comprising non-bacterial PUFA PKSsystems, to methods of making and using such systems for the productionof products of interest, including bioactive molecules, and to novelmethods for identifying new eukaryotic microorganisms having such a PUFAPKS system. As used herein, a PUFA PKS system generally has thefollowing identifying features: (1) it produces PUFAs as a naturalproduct of the system; and (2) it comprises several multifunctionalproteins assembled into a complex that conducts both iterativeprocessing of the fatty acid chain as well non-iterative processing,including trans-cis isomerization and enoyl reduction reactions inselected cycles (See FIG. 1, for example).

More specifically, first, a PUFA PKS system that forms the basis of thisinvention produces polyunsaturated fatty acids (PUFAs) as products(i.e., an organism that endogenously (naturally) contains such a PKSsystem makes PUFAs using this system). The PUFAs referred to herein arepreferably polyunsaturated fatty acids with a carbon chain length of atleast 16 carbons, and more preferably at least 18 carbons, and morepreferably at least 20 carbons, and more preferably 22 or more carbons,with at least 3 or more double bonds, and preferably 4 or more, and morepreferably 5 or more, and even more preferably 6 or more double bonds,wherein all double bonds are in the cis configuration. It is an objectof the present invention to find or create via genetic manipulation ormanipulation of the endproduct, PKS systems which producepolyunsaturated fatty acids of desired chain length and with desirednumbers of double bonds. Examples of PUFAs include, but are not limitedto, DHA (docosahexaenoic acid (C22:6, ω-3)), DPA (docosapentaenoic acid(C22:5, ω-6)), and EPA (eicosapentaenoic acid (C20:5, ω-3)).

Second, the PUFA PKS system described herein incorporates both iterativeand non-iterative reactions, which distinguish the system frompreviously described PKS systems (e.g., type I, type II or modular).More particularly, the PUFA PKS system described herein contains domainsthat appear to function during each cycle as well as those which appearto function during only some of the cycles. A key aspect of this may berelated to the domains showing homology to the bacterial Fab A enzymes.For example, the Fab A enzyme of E. coli has been shown to possess twoenzymatic activities. It possesses a dehydration activity in which awater molecule (H₂O) is abstracted from a carbon chain containing ahydroxy group, leaving a trans double bond in that carbon chain. Inaddition, it has an isomerase activity in which the trans double bond isconverted to the cis configuration. This isomerization is accomplishedin conjunction with a migration of the double bond position to adjacentcarbons. In PKS (and FAS) systems, the main carbon chain is extended in2 carbon increments. One can therefore predict the number of extensionreactions required to produce the PUFA products of these PKS systems.For example, to produce DHA (C22:6, all cis) requires 10 extensionreactions. Since there are only 6 double bonds in the end product, itmeans that during some of the reaction cycles, a double bond is retained(as a cis isomer), and in others, the double bond is reduced prior tothe next extension.

Before the discovery of a PUFA PKS system in marine bacteria (see U.S.Pat. No. 6,140,486), PKS systems were not known to possess thiscombination of iterative and selective enzymatic reactions, and theywere not thought of as being able to produce carbon-carbon double bondsin the cis configuration. However, the PUFA PKS system described by thepresent invention has the capacity to introduce cis double bonds and thecapacity to vary the reaction sequence in the cycle.

Therefore, the present inventors propose to use these features of thePUFA PKS system to produce a range of bioactive molecules that could notbe produced by the previously described (Type II, Type I and modular)PKS systems. These bioactive molecules include, but are limited to,polyunsaturated fatty acids (PUFAs), antibiotics or other bioactivecompounds, many of which will be discussed below. For example, using theknowledge of the PUFA PKS gene structures described herein, any of anumber of methods can be used to alter the PUFA PKS genes, or combineportions of these genes with other synthesis systems, including otherPKS systems, such that new products are produced. The inherent abilityof this particular type of system to do both iterative and selectivereactions will enable this system to yield products that would not befound if similar methods were applied to other types of PKS systems.

In one embodiment, a PUFA PKS system according to the present inventioncomprises at least the following biologically active domains: (a) atleast two enoyl ACP-reductase (ER) domains; (b) at least six acylcarrier protein (ACP) domains; (c) at least two β-keto acyl-ACP synthase(KS) domains; (d) at least one acyltransferase (AT) domain; (e) at leastone ketoreductase (KR) domain; (f) at least two FabA-like β-hydroxyacyl-ACP dehydrase (DH) domains; (g) at least one chain length factor(CLF) domain; and (h) at least one malonyl-CoA:ACP acyltransferase (MAT)domain. The functions of these domains are generally individually knownin the art and will be described in detail below with regard to the PUFAPKS system of the present invention.

In another embodiment, the PUFA PKS system comprises at least thefollowing biologically active domains: (a) at least one enoylACP-reductase (ER) domain; (b) multiple acyl carrier protein (ACP)domains (at least four, and preferably at least five, and morepreferably at least six, and even more preferably seven, eight, nine, ormore than nine); (c) at least two β-keto acyl-ACP synthase (KS) domains;(d) at least one acyltransferase (AT) domain; (e) at least oneketoreductase (KR) domain; (f) at least two FabA-like β-hydroxy acyl-ACPdehydrase (DH) domains; (g) at least one chain length factor (CLF)domain; and (h) at least one malonyl-CoA:ACP acyltransferase (MAT)domain. Preferably, such a PUFA PKS system is a non-bacterial PUFA-PKSsystem.

In one embodiment, a PUFA PKS system of the present invention is anon-bacterial PUFA PKS system. In other words, in one embodiment, thePUFA PKS system of the present invention is isolated from an organismthat is not a bacteria, or is a homologue of or derived from a PUFA PKSsystem from an organism that is not a bacteria, such as a eukaryote oran archaebacterium. Eukaryotes are separated from prokaryotes based onthe degree of differentiation of the cells. The higher group with moredifferentiation is called eukaryotic. The lower group with lessdifferentiated cells is called prokaryotic. In general, prokaryotes dono possess a nuclear membrane, do not exhibit mitosis during celldivision, have only one chromosome, their cytoplasm contains 70Sribosomes, they do not possess any mitochondria, endoplasmic reticulum,chloroplasts, lysosomes or golgi apparatus, their flagella (if present)consists of a single fibril. In contrast eukaryotes have a nuclearmembrane, they do exhibit mitosis during cell division, they have manychromosomes, their cytoplasm contains 80S ribosomes, they do possessmitochondria, endoplasmic reticulum, chloroplasts (in algae), lysosomesand golgi apparatus, and their flagella (if present) consists of manyfibrils. In general, bacteria are prokaryotes, while algae, fungi,protist, protozoa and higher plants are eukaryotes. The PUFA PKS systemsof the marine bacteria (e.g., Shewanella and Vibrio marinus) are not thebasis of the present invention, although the present invention doescontemplate the use of domains from these bacterial PUFA PKS systems inconjunction with domains from the non-bacterial PUFA PKS systems of thepresent invention. For example, according to the present invention,genetically modified organisms can be produced which incorporatenon-bacterial PUFA PKS functional domains with bacteria PUFA PKSfunctional domains, as well as PKS functional domains or proteins fromother PKS systems (type I, type II, modular) or FAS systems.

Schizochytrium is a Thraustochytrid marine microorganism thataccumulates large quantities of triacylglycerols rich in DHA anddocosapentaenoic acid (DPA; 22:5 ω-6); e.g., 30% DHA+DPA by dry weight(Barclay et al., J. Appl. Phycol. 6, 123 (1994)). In eukaryotes thatsynthesize 20- and 22-carbon PUFAs by an elongation/desaturationpathway, the pools of 18-, 20- and 22-carbon intermediates arerelatively large so that in vivo labeling experiments using[¹⁴C]-acetate reveal clear precursor-product kinetics for the predictedintermediates (Gellerman et al., Biochim. Biophys. Acta 573:23 (1979)).Furthermore, radiolabeled intermediates provided exogenously to suchorganisms are converted to the final PUFA products. The presentinventors have shown that [1-¹⁴C]-acetate was rapidly taken up bySchizochytrium cells and incorporated into fatty acids, but at theshortest labeling time (1 min), DHA contained 31% of the label recoveredin fatty acids, and this percentage remained essentially unchangedduring the 10-15 min of [¹⁴C]-acetate incorporation and the subsequent24 hours of culture growth (See Example 3). Similarly, DPA represented10% of the label throughout the experiment. There is no evidence for aprecursor-product relationship between 16- or 18-carbon fatty acids andthe 22-carbon polyunsaturated fatty acids. These results are consistentwith rapid synthesis of DHA from [¹⁴C]-acetate involving very small(possibly enzyme-bound) pools of intermediates. A cell-free homogenatederived from Schizochytrium cultures incorporated [1-¹⁴C]-malonyl-CoAinto DHA, DPA, and saturated fatty acids. The same biosyntheticactivities were retained by a 100,000×g supernatant fraction but werenot present in the membrane pellet. Thus, DHA and DPA synthesis inSchizochytrium does not involve membrane-bound desaturases or fatty acidelongation enzymes like those described for other eukaryotes(Parker-Barnes et al., 2000, supra; Shanklin et al., 1998, supra). Thesefractionation data contrast with those obtained from the Shewanellaenzymes (See Metz et al., 2001, supra) and may indicate use of adifferent (soluble) acyl acceptor molecule, such as CoA, by theSchizochytrium enzyme.

In copending U.S. application Ser. No. 09/231,899, a cDNA library fromSchizochytrium was constructed and approximately 8,000 random clones(ESTs) were sequenced. Within this dataset, only one moderatelyexpressed gene (0.3% of all sequences) was identified as a fatty aciddesaturase, although a second putative desaturase was represented by asingle clone (0.01%). By contrast, sequences that exhibited homology to8 of the 11 domains of the Shewanella PKS genes shown in FIG. 2 were allidentified at frequencies of 0.2-0.5%. In U.S. application Ser. No.09/231,899, several cDNA clones showing homology to the Shewanella PKSgenes were sequenced, and various clones were assembled into nucleicacid sequences representing two partial open reading frames and onecomplete open reading frame. Nucleotides 390-4443 of the cDNA sequencecontaining the first partial open reading frame described in U.S.application Ser. No. 09/231,899 (denoted therein as SEQ ID NO:69) matchnucleotides 4677-8730 (plus the stop codon) of the sequence denotedherein as OrfA (SEQ ID NO:1). Nucleotides 1-4876 of the cDNA sequencecontaining the second partial open reading frame described in U.S.application Ser. No. 09/231,899 (denoted therein as SEQ ID NO:71)matches nucleotides 1311-6177 (plus the stop codon) of the sequencedenoted herein as OrfB (SEQ ID NO:3). Nucleotides 145-4653 of the cDNAsequence containing the complete open reading frame described in U.S.application Ser. No. 09/231,899 (denoted therein as SEQ ID NO:76 andincorrectly designated as a partial open reading frame) match the entiresequence (plus the stop codon) of the sequence denoted herein as OrfC(SEQ ID NO:5).

Further sequencing of cDNA and genomic clones by the present inventorsallowed the identification of the full-length genomic sequence of eachof OrfA, OrfB and OrfC and the complete identification of the domainswith homology to those in Shewanella (see FIG. 2). It is noted that inSchizochytrium, the genomic DNA and cDNA are identical, due to the lackof introns in the organism genome, to the best of the present inventors'knowledge. Therefore, reference to a nucleotide sequence fromSchizochytrium can refer to genomic DNA or cDNA. Based on the comparisonof the Schizochytrium PKS domains to Shewanella, clearly, theSchizochytrium genome encodes proteins that are highly similar to theproteins in Shewanella that are capable of catalyzing EPA synthesis. Theproteins in Schizochytrium constitute a PUFA PKS system that catalyzesDHA and DPA synthesis. As discussed in detail herein, simplemodification of the reaction scheme identified for Shewanella will allowfor DHA synthesis in Schizochytrium. The homology between theprokaryotic Shewanella and eukaryotic Schizochytrium genes suggests thatthe PUFA PKS has undergone lateral gene transfer.

FIG. 1 is a graphical representation of the three open reading framesfrom the Schizochytrium PUFA PKS system, and includes the domainstructure of this PUFA PKS system. As described in Example 1 below, thedomain structure of each open reading frame is as follows:

Open Reading Frame A (OrfA):

The complete nucleotide sequence for OrfA is represented herein as SEQID NO:1. Nucleotides 4677-8730 of SEQ ID NO:1 correspond to nucleotides390-4443 of the sequence denoted as SEQ ID NO:69 in U.S. applicationSer. No. 09/231,899. Therefore, nucleotides 1-4676 of SEQ ID NO:1represent additional sequence that was not disclosed in U.S. applicationSer. No. 09/231,899. This novel region of SEQ ID NO:1 encodes thefollowing domains in OrfA: (1) the ORFA-KS domain; (2) the ORFA-MATdomain; and (3) at least a portion of the ACP domain region (e.g., atleast ACP domains 1-4). It is noted that nucleotides 1-389 of SEQ IDNO:69 in U.S. application Ser. No. 09/231,899 do not match with the 389nucleotides that are upstream of position 4677 in SEQ ID NO:1 disclosedherein. Therefore, positions 1-389 of SEQ ID NO:69 in U.S. applicationSer. No. 09/231,899 appear to be incorrectly placed next to nucleotides390-4443 of that sequence. Most of these first 389 nucleotides (aboutpositions 60-389) are a match with an upstream portion of OrfA (SEQ IDNO:1) of the present invention and therefore, it is believed that anerror occurred in the effort to prepare the contig of the cDNAconstructs in U.S. application Ser. No. 09/231,899. The region in whichthe alignment error occurred in U.S. application Ser. No. 09/231,899 iswithin the region of highly repetitive sequence (i.e., the ACP region,discussed below), which probably created some confusion in the assemblyof that sequence from various cDNA clones.

OrfA is a 8730 nucleotide sequence (not including the stop codon) whichencodes a 2910 amino acid sequence, represented herein as SEQ ID NO:2.Within OrfA are twelve domains: (a) one β-keto acyl-ACP synthase (KS)domain; (b) one malonyl-CoA:ACP acyltransferase (MAT) domain; (c) nineacyl carrier protein (ACP) domains; and (d) one ketoreductase (KR)domain.

The nucleotide sequence for OrfA has been deposited with GenBank asAccession No. AF378327 (amino acid sequence Accession No. AAK728879).OrfA was compared with known sequences in a standard BLAST search (BLAST2.0 Basic BLAST homology search using blastp for amino acid searches,blastn for nucleic acid searches, and blastX for nucleic acid searchesand searches of the translated amino acid sequence in all 6 open readingframes with standard default parameters, wherein the query sequence isfiltered for low complexity regions by default (described in Altschul,S. F., Madden, T. L., Schääffer, A. A., Zhang, J., Zhang, Z., Miller, W.& Lipman, D. J. (1997) “Gapped BLAST and PSI-BLAST: a new generation ofprotein database search programs.” Nucleic Acids Res. 25:3389-3402,incorporated herein by reference in its entirety)). At the nucleic acidlevel, OrfA has no significant homology to any known nucleotidesequence. At the amino acid level, the sequences with the greatestdegree of homology to ORFA were: Nostoc sp. 7120 heterocyst glycolipidsynthase (Accession No. NC_(—)003272), which was 42% identical to ORFAover 1001 amino acid residues; and Moritella marinus (Vibrio marinus)ORF8 (Accession No. AB025342), which was 40% identical to ORFA over 993amino acid residues.

The first domain in OrfA is a KS domain, also referred to herein asORFA-KS. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1 and 40 ofSEQ ID NO:1 (OrfA) to an ending point of between about positions 1428and 1500 of SEQ ID NO:1. The nucleotide sequence containing the sequenceencoding the ORFA-KS domain is represented herein as SEQ ID NO:7(positions 1-1500 of SEQ ID NO:1). The amino acid sequence containingthe KS domain spans from a starting point of between about positions 1and 14 of SEQ ID NO:2 (ORFA) to an ending point of between aboutpositions 476 and 500 of SEQ ID NO:2. The amino acid sequence containingthe ORFA-KS domain is represented herein as SEQ ID NO:8 (positions 1-500of SEQ ID NO:2). It is noted that the ORFA-KS domain contains an activesite motif. DXAC* (*acyl binding site C₂₁₅).

According to the present invention, a domain or protein having 3-ketoacyl-ACP synthase (KS) biological activity (function) is characterizedas the enzyme that carries out the initial step of the FAS (and PKS)elongation reaction cycle. The acyl group destined for elongation islinked to a cysteine residue at the active site of the enzyme by athioester bond. In the multi-step reaction, the acyl-enzyme undergoescondensation with malonyl-ACP to form -keto acyl-ACP, CO₂ and freeenzyme. The KS plays a key role in the elongation cycle and in manysystems has been shown to possess greater substrate specificity thanother enzymes of the reaction cycle. For example, E. coli has threedistinct KS enzymes—each with its own particular role in the physiologyof the organism (Magnuson et al., Microbiol. Rev. 57, 522 (1993)). Thetwo KS domains of the PUFA-PKS systems could have distinct roles in thePUFA biosynthetic reaction sequence.

As a class of enzymes, KS's have been well characterized. The sequencesof many verified KS genes are know, the active site motifs have beenidentified and the crystal structures of several have been determined.Proteins (or domains of proteins) can be readily identified as belongingto the KS family of enzymes by homology to known KS sequences.

The second domain in OrfA is a MAT domain, also referred to herein asORFA-MAT. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1723 and 1798of SEQ ID NO:1 (OrfA) to an ending point of between about positions 2805and 3000 of SEQ ID NO:1. The nucleotide sequence containing the sequenceencoding the ORFA-MAT domain is represented herein as SEQ ID NO:9(positions 1723-3000 of SEQ ID NO:1). The amino acid sequence containingthe MAT domain spans from a starting point of between about positions575 and 600 of SEQ ID NO:2 (ORFA) to an ending point of between aboutpositions 935 and 1000 of SEQ ID NO:2. The amino acid sequencecontaining the ORFA-MAT domain is represented herein as SEQ ID NO:10(positions 575-1000 of SEQ ID NO:2). It is noted that the ORFA-MATdomain contains an active site motif: GHS*XG (*acyl binding site S₇₀₆),represented herein as SEQ ID NO: 11.

According to the present invention, a domain or protein havingmalonyl-CoA:ACP acyltransferase (MAT) biological activity (function) ischaracterized as one that transfers the malonyl moiety from malonyl-CoAto ACP. In addition to the active site motif (GxSxG), these enzymespossess an extended motif® and Q amino acids in key positions) thatidentifies them as MAT enzymes (in contrast to the AT domain ofSchizochytrium Orf B). In some PKS systems (but not the PUFA PKS domain)MAT domains will preferentially load methyl- or ethyl-malonate on to theACP group (from the corresponding CoA ester), thereby introducingbranches into the linear carbon chain. MAT domains can be recognized bytheir homology to known MAT sequences and by their extended motifstructure.

Domains 3-11 of OrfA are nine tandem ACP domains, also referred toherein as ORFA-ACP (the first domain in the sequence is ORFA-ACP 1, thesecond domain is ORFA-ACP2, the third domain is ORFA-ACP3, etc.). Thefirst ACP domain, ORFA-ACP1, is contained within the nucleotide sequencespanning from about position 3343 to about position 3600 of SEQ ID NO:1(OrfA). The nucleotide sequence containing the sequence encoding theORFA-ACP1 domain is represented herein as SEQ ID NO:12 (positions3343-3600 of SEQ ID NO:1). The amino acid sequence containing the firstACP domain spans from about position 1115 to about position 1200 of SEQID NO:2. The amino acid sequence containing the ORFA-ACP1 domain isrepresented herein as SEQ ID NO:13 (positions 1115-1200 of SEQ ID NO:2).It is noted that the ORFA-ACP1 domain contains an active site motif:LGIDS* (*pantetheine binding motif S₁₁₅₇), represented herein by SEQ IDNO:14.

The nucleotide and amino acid sequences of all nine ACP domains arehighly conserved and therefore, the sequence for each domain is notrepresented herein by an individual sequence identifier. However, basedon the information disclosed herein, one of skill in the art can readilydetermine the sequence containing each of the other eight ACP domains(see discussion below).

All nine ACP domains together span a region of OrfA of from aboutposition 3283 to about position 6288 of SEQ ID NO:1, which correspondsto amino acid positions of from about 1095 to about 2096 of SEQ ID NO:2.The nucleotide sequence for the entire ACP region containing all ninedomains is represented herein as SEQ ID NO:16. The region represented bySEQ ID NO:16 includes the linker segments between individual ACPdomains. The repeat interval for the nine domains is approximately every330 nucleotides of SEQ ID NO:16 (the actual number of amino acidsmeasured between adjacent active site serines ranges from 104 to 116amino acids). Each of the nine ACP domains contains a pantetheinebinding motif LGIDS* (represented herein by SEQ ID NO:14), wherein S* isthe pantetheine binding site serine (S). The pantetheine binding siteserine (S) is located near the center of each ACP domain sequence. Ateach end of the ACP domain region and between each ACP domain is aregion that is highly enriched for proline (P) and alanine (A), which isbelieved to be a linker region. For example, between ACP domains 1 and 2is the sequence: APAPVKAAAPAAPVASAPAPA, represented herein as SEQ IDNO:15. The locations of the active site serine residues (i.e., thepantetheine binding site) for each of the nine ACP domains, with respectto the amino acid sequence of SEQ ID NO:2, are as follows: ACP1=S₁₁₅₇;ACP2=S₁₂₆₆; ACP3=S₁₃₇₇; ACP4=S₁₄₈₈; ACP5=S₁₆₀₄; ACP6=S₁₇₁₅; ACP7=S₁₈₁₉;ACP8=S₁₉₃₀; and ACP9=S₂₀₃₄. Given that the average size of an ACP domainis about 85 amino acids, excluding the linker, and about 110 amino acidsincluding the linker, with the active site serine being approximately inthe center of the domain, one of skill in the art can readily determinethe positions of each of the nine ACP domains in OrfA.

According to the present invention, a domain or protein having acylcarrier protein (ACP) biological activity (function) is characterized asbeing small polypeptides (typically, 80 to 100 amino acids long), thatfunction as carriers for growing fatty acyl chains via a thioesterlinkage to a covalently bound co-factor of the protein. They occur asseparate units or as domains within larger proteins. ACPs are convertedfrom inactive apo-forms to functional holo-forms by transfer of thephosphopantetheinyl moeity of CoA to a highly conserved serine residueof the ACP. Acyl groups are attached to ACP by a thioester linkage atthe free terminus of the phosphopantetheinyl moiety. ACPs can beidentified by labeling with radioactive pantetheine and by sequencehomology to known ACPs. The presence of variations of the abovementioned motif (LGIDS*) is also a signature of an ACP.

Domain 12 in OrfA is a KR domain, also referred to herein as ORFA-KR.This domain is contained within the nucleotide sequence spanning from astarting point of about position 6598 of SEQ ID NO:1 to an ending pointof about position 8730 of SEQ ID NO:1. The nucleotide sequencecontaining the sequence encoding the ORFA-KR domain is representedherein as SEQ ID NO:17 (positions 6598-8730 of SEQ ID NO:1). The aminoacid sequence containing the KR domain spans from a starting point ofabout position 2200 of SEQ ID NO:2 (ORFA) to an ending point of aboutposition 2910 of SEQ ID NO:2. The amino acid sequence containing theORFA-KR domain is represented herein as SEQ ID NO:18 (positions2200-2910 of SEQ ID NO:2). Within the KR domain is a core region withhomology to short chain aldehyde-dehydrogenases (KR is a member of thisfamily). This core region spans from about position 7198 to aboutposition 7500 of SEQ ID NO:1, which corresponds to amino acid positions2400-2500 of SEQ ID NO:2.

According to the present invention, a domain or protein havingketoreductase activity, also referred to as 3-ketoacyl-ACP reductase(KR) biological activity (function), is characterized as one thatcatalyzes the pyridine-nucleotide-dependent reduction of 3-keto acylforms of ACP. It is the first reductive step in the de novo fatty acidbiosynthesis elongation cycle and a reaction often performed inpolyketide biosynthesis. Significant sequence similarity is observedwith one family of enoyl ACP reductases (ER), the other reductase of FAS(but not the ER family present in the PUFA PKS system), and theshort-chain alcohol dehydrogenase family. Pfam analysis of the PUFA PKSregion indicated above reveals the homology to the short-chain alcoholdehydrogenase family in the core region. Blast analysis of the sameregion reveals matches in the core area to known KR enzymes as well asan extended region of homology to domains from the other characterizedPUFA PKS systems.

Open Reading Frame B (OrfB):

The complete nucleotide sequence for OrfB is represented herein as SEQID NO:3. Nucleotides 1311-4242 and 4244-6177 of SEQ ID NO:3 correspondto nucleotides 1-2932 and 2934-4867 of the sequence denoted as SEQ IDNO:71 in U.S. application Ser. No. 09/231,899 (The cDNA sequence in U.S.application Ser. No. 09/231,899 contains about 345 additionalnucleotides beyond the stop codon, including a polyA tail). Therefore,nucleotides 1-1310 of SEQ ID NO:1 represent additional sequence that wasnot disclosed in U.S. application Ser. No. 09/231,899. This novel regionof SEQ ID NO:3 contains most of the KS domain encoded by OrfB.

OrfB is a 6177 nucleotide sequence (not including the stop codon) whichencodes a 2059 amino acid sequence, represented herein as SEQ ID NO:4.Within OrfB are four domains: (a) one β-keto acyl-ACP synthase (KS)domain; (b) one chain length factor (CLF) domain; (c) one acyltransferase (AT) domain; and, (d) one enoyl ACP-reductase (ER) domain.

The nucleotide sequence for OrfB has been deposited with GenBank asAccession No. AF378328 (amino acid sequence Accession No. AAK728880).OrfB was compared with known sequences in a standard BLAST search asdescribed above. At the nucleic acid level, OrfB has no significanthomology to any known nucleotide sequence. At the amino acid level, thesequences with the greatest degree of homology to ORFB were: Shewanellasp. hypothetical protein (Accession No. U73935), which was 53% identicalto ORFB over 458 amino acid residues; Moritella marinus (Vibrio marinus)ORF11 (Accession No. AB025342), which was 53% identical to ORFB over 460amino acid residues; Photobacterium profundum omega-3 polyunsaturatedfatty acid synthase PfaD (Accession No. AF409100), which was 52%identical to ORFB over 457 amino acid residues; and Nostoc sp. 7120hypothetical protein (Accession No. NC_(—)003272), which was 53%identical to ORFB over 430 amino acid residues.

The first domain in OrfB is a KS domain, also referred to herein asORFB-KS. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1 and 43 ofSEQ ID NO:3 (OrfB) to an ending point of between about positions 1332and 1350 of SEQ ID NO:3. The nucleotide sequence containing the sequenceencoding the ORFB-KS domain is represented herein as SEQ ID NO:19(positions 1-1350 of SEQ ID NO:3). The amino acid sequence containingthe KS domain spans from a starting point of between about positions 1and 15 of SEQ ID NO:4 (ORFB) to an ending point of between aboutpositions 444 and 450 of SEQ ID NO:4. The amino acid sequence containingthe ORFB-KS domain is represented herein as SEQ ID NO:20 (positions1-450 of SEQ ID NO:4). It is noted that the ORFB-KS domain contains anactive site motif: DXAC* (*acyl binding site C₁₉₆). KS biologicalactivity and methods of identifying proteins or domains having suchactivity is described above.

The second domain in OrfB is a CLF domain, also referred to herein asORFB-CLF. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1378 and 1402of SEQ ID NO:3 (OrfB) to an ending point of between about positions 2682and 2700 of SEQ ID NO:3. The nucleotide sequence containing the sequenceencoding the ORFB-CLF domain is represented herein as SEQ ID NO:21(positions 1378-2700 of SEQ ID NO:3). The amino acid sequence containingthe CLF domain spans from a starting point of between about positions460 and 468 of SEQ ID NO:4 (ORFB) to an ending point of between aboutpositions 894 and 900 of SEQ ID NO:4. The amino acid sequence containingthe ORFB-CLF domain is represented herein as SEQ ID NO:22 (positions460-900 of SEQ ID NO:4). It is noted that the ORFB-CLF domain contains aKS active site motif without the acyl-binding cysteine.

According to the present invention, a domain or protein is referred toas a chain length factor (CLF) based on the following rationale. The CLFwas originally described as characteristic of Type II (dissociatedenzymes) PKS systems and was hypothesized to play a role in determiningthe number of elongation cycles, and hence the chain length, of the endproduct. CLF amino acid sequences show homology to KS domains (and arethought to form heterodimers with a KS protein), but they lack theactive site cysteine. CLF's role in PKS systems is currentlycontroversial. New evidence (C. Bisang et al., Nature 401, 502 (1999))suggests a role in priming (providing the initial acyl group to beelongated) the PKS systems. In this role the CLF domain is thought todecarboxylate malonate (as malonyl-ACP), thus forming an acetate groupthat can be transferred to the KS active site. This acetate thereforeacts as the ‘priming’ molecule that can undergo the initial elongation(condensation) reaction. Homologues of the Type II CLF have beenidentified as ‘loading’ domains in some modular PKS systems. A domainwith the sequence features of the CLF is found in all currentlyidentified PUFA PKS systems and in each case is found as part of amultidomain protein.

The third domain in OrfB is an AT domain, also referred to herein asORFB-AT. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 2701 and 3598of SEQ ID NO:3 (OrfB) to an ending point of between about positions 3975and 4200 of SEQ ID NO:3. The nucleotide sequence containing the sequenceencoding the ORFB-AT domain is represented herein as SEQ ID NO:23(positions 2701-4200 of SEQ ID NO:3). The amino acid sequence containingthe AT domain spans from a starting point of between about positions 901and 1200 of SEQ ID NO:4 (ORFB) to an ending point of between aboutpositions 1325 and 1400 of SEQ ID NO:4. The amino acid sequencecontaining the ORFB-AT domain is represented herein as SEQ ID NO:24(positions 901-1400 of SEQ ID NO:4). It is noted that the ORFB-AT domaincontains an active site motif of GxS*xG (*acyl binding site S₁₁₄₀) thatis characteristic of acyltransferse (AT) proteins.

An “acyltransferase” or “AT” refers to a general class of enzymes thatcan carry out a number of distinct acyl transfer reactions. TheSchizochytrium domain shows good homology to a domain present in all ofthe other PUFA PKS systems currently examined and very weak homology tosome acyltransferases whose specific functions have been identified(e.g. to malonyl-CoA:ACP acyltransferase, MAT). In spite of the weakhomology to MAT, this AT domain is not believed to function as a MATbecause it does not possess an extended motif structure characteristicof such enzymes (see MAT domain description, above). For the purposes ofthis disclosure, the functions of the AT domain in a PUFA PKS systeminclude, but are not limited to: transfer of the fatty acyl group fromthe ORFA ACP domain(s) to water (i.e. a thioesterase—releasing the fattyacyl group as a free fatty acid), transfer of a fatty acyl group to anacceptor such as CoA, transfer of the acyl group among the various ACPdomains, or transfer of the fatty acyl group to a lipophilic acceptormolecule (e.g. to lysophosphadic acid).

The fourth domain in OrfB is an ER domain, also referred to herein asORFB-ER. This domain is contained within the nucleotide sequencespanning from a starting point of about position 4648 of SEQ ID NO:3(OrfB) to an ending point of about position 6177 of SEQ ID NO:3. Thenucleotide sequence containing the sequence encoding the ORFB-ER domainis represented herein as SEQ ID NO:25 (positions 4648-6177 of SEQ IDNO:3). The amino acid sequence containing the ER domain spans from astarting point of about position 1550 of SEQ ID NO:4 (ORFB) to an endingpoint of about position 2059 of SEQ ID NO:4. The amino acid sequencecontaining the ORFB-ER domain is represented herein as SEQ ID NO:26(positions 1550-2059 of SEQ ID NO:4).

According to the present invention, this domain has enoyl reductase (ER)biological activity. The ER enzyme reduces the trans-double bond(introduced by the DH activity) in the fatty acyl-ACP, resulting infully saturating those carbons. The ER domain in the PUFA-PKS showshomology to a newly characterized family of ER enzymes (Heath et al.,Nature 406, 145 (2000)). Heath and Rock identified this new class of ERenzymes by cloning a gene of interest from Streptococcus pneumoniae,purifying a protein expressed from that gene, and showing that it had ERactivity in an in vitro assay. The sequence of the Schizochytrium ERdomain of OrfB shows homology to the S. pneumoniae ER protein. All ofthe PUFA PKS systems currently examined contain at least one domain withvery high sequence homology to the Schizochytrium ER domain. TheSchizochytrium PUFA PKS system contains two ER domains (one on OrfB andone on OrfC).

Open Reading Frame C (OrfC):

The complete nucleotide sequence for OrfC is represented herein as SEQID NO:5. Nucleotides 1-4506 of SEQ ID NO:5 (i.e., the entire openreading frame sequence, not including the stop codon) correspond tonucleotides 145-2768, 2770-2805, 2807-2817, and 2819-4653 of thesequence denoted as SEQ ID NO:76 in U.S. application Ser. No. 09/231,899(The cDNA sequence in U.S. application Ser. No. 09/231,899 containsabout 144 nucleotides upstream of the start codon for OrfC and about 110nucleotides beyond the stop codon, including a polyA tail). OrfC is a4506 nucleotide sequence (not including the stop codon) which encodes a1502 amino acid sequence, represented herein as SEQ ID NO:6. Within OrfCare three domains: (a) two FabA-like β-hydroxy acyl-ACP dehydrase (DH)domains; and (b) one enoyl ACP-reductase (ER) domain.

The nucleotide sequence for OrfC has been deposited with GenBank asAccession No. AF378329 (amino acid sequence Accession No. AAK728881).OrfC was compared with known sequences in a standard BLAST search asdescribed above. At the nucleic acid level, OrfC has no significanthomology to any known nucleotide sequence. At the amino acid level(Blastp), the sequences with the greatest degree of homology to ORFCwere: Moritella marinus (Vibrio marinus) ORF11 (Accession No. ABO25342),which is 45% identical to ORFC over 514 amino acid residues, Shewanellasp. hypothetical protein 8 (Accession No. U73935), which is 49%identical to ORFC over 447 amino acid residues, Nostoc sp. hypotheticalprotein (Accession No. NC_(—)003272), which is 49% identical to ORFCover 430 amino acid residues, and Shewanella sp. hypothetical protein 7(Accession No. U73935), which is 37% identical to ORFC over 930 aminoacid residues.

The first domain in OrfC is a DH domain, also referred to herein asORFC-DH1. This is one of two DH domains in OrfC, and therefore isdesignated DH1. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1 and 778 ofSEQ ID NO:5 (OrfC) to an ending point of between about positions 1233and 1350 of SEQ ID NO:5. The nucleotide sequence containing the sequenceencoding the ORFC-DH1 domain is represented herein as SEQ ID NO:27(positions 1-1350 of SEQ ID NO:5). The amino acid sequence containingthe DH1 domain spans from a starting point of between about positions 1and 260 of SEQ ID NO:6 (ORFC) to an ending point of between aboutpositions 411 and 450 of SEQ ID NO:6. The amino acid sequence containingthe ORFC-DH1 domain is represented herein as SEQ ID NO:28 (positions1-450 of SEQ ID NO:6).

The characteristics of both the DH domains (see below for DH 2) in thePUFA PKS systems have been described in the preceding sections. Thisclass of enzyme removes HOH from a β-keto acyl-ACP and leaves a transdouble bond in the carbon chain. The DH domains of the PUFA PKS systemsshow homology to bacterial DH enzymes associated with their FAS systems(rather than to the DH domains of other PKS systems). A subset ofbacterial DH's, the FabA-like DH's, possesses cis-trans isomeraseactivity (Heath et al., J. Biol. Chem., 271, 27795 (1996)). It is thehomologies to the FabA-like DH's that indicate that one or both of theDH domains is responsible for insertion of the cis double bonds in thePUFA PKS products.

The second domain in OrfC is a DH domain, also referred to herein asORFC-DH2. This is the second of two DH domains in OrfC, and therefore isdesignated DH2. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1351 and 2437of SEQ ID NO :5 (OrfC) to an ending point of between about positions2607 and 2847 of SEQ ID NO:5. The nucleotide sequence containing thesequence encoding the ORFC-DH2 domain is represented herein as SEQ IDNO:29 (positions 1351-2847 of SEQ ID NO:5). The amino acid sequencecontaining the DH2 domain spans from a starting point of between aboutpositions 451 and 813 of SEQ ID NO :6 (ORFC) to an ending point ofbetween about positions 869 and 949 of SEQ ID NO:6. The amino acidsequence containing the ORFC-DH2 domain is represented herein as SEQ IDNO:30 (positions 451-949 of SEQ ID NO:6). DH biological activity hasbeen described above.

The third domain in OrfC is an ER domain, also referred to herein asORFC-ER. This domain is contained within the nucleotide sequencespanning from a starting point of about position 2995 of SEQ ID NO:5(OrfC) to an ending point of about position 4506 of SEQ ID NO:5. Thenucleotide sequence containing the sequence encoding the ORFC-ER domainis represented herein as SEQ ID NO:31 (positions 2995-4506 of SEQ IDNO:5). The amino acid sequence containing the ER domain spans from astarting point of about position 999 of SEQ ID NO:6 (ORFC) to an endingpoint of about position 1502 of SEQ ID NO:6. The amino acid sequencecontaining the ORFC-ER domain is represented herein as SEQ ID NO:32(positions 999-1502 of SEQ ID NO:6). ER biological activity has beendescribed above.

One embodiment of the present invention relates to an isolated nucleicacid molecule comprising a nucleic acid sequence from a non-bacterialPUFA PKS system, a homologue thereof, a fragment thereof, and/or anucleic acid sequence that is complementary to any of such nucleic acidsequences. In one aspect, the present invention relates to an isolatednucleic acid molecule comprising a nucleic acid sequence selected fromthe group consisting of: (a) a nucleic acid sequence encoding an aminoacid sequence selected from the group consisting of: SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO: 6, and biologically active fragments thereof, (b) anucleic acid sequence encoding an amino acid sequence selected from thegroup consisting of: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, and biologically active fragmentsthereof; (c) a nucleic acid sequence encoding an amino acid sequencethat is at least about 60% identical to at least 500 consecutive aminoacids of said amino acid sequence of (a), wherein said amino acidsequence has a biological activity of at least one domain of apolyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system; (d)a nucleic acid sequence encoding an amino acid sequence that is at leastabout 60% identical to said amino acid sequence of (b), wherein saidamino acid sequence has a biological activity of at least one domain ofa polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system; or(e) a nucleic acid sequence that is fully complementary to the nucleicacid sequence of (a), (b), (c), or (d). In a further embodiment, nucleicacid sequences including a sequence encoding the active site domains orother functional motifs described above for several of the PUFA PKSdomains are encompassed by the invention.

According to the present invention, an amino acid sequence that has abiological activity of at least one domain of a PUFA PKS system is anamino acid sequence that has the biological activity of at least onedomain of the PUFA PKS system described in detail herein, as exemplifiedby the Schizochytrium PUFA PKS system. The biological activities of thevarious domains within the Schizochytrium PUFA PKS system have beendescribed in detail above. Therefore, an isolated nucleic acid moleculeof the present invention can encode the translation product of any PUFAPKS open reading frame, PUFA PKS domain, biologically active fragmentthereof, or any homologue of a naturally occurring PUFA PKS open readingframe or domain which has biological activity. A homologue of givenprotein or domain is a protein or polypeptide that has an amino acidsequence which differs from the naturally occurring reference amino acidsequence (i.e., of the reference protein or domain) in that at least oneor a few, but not limited to one or a few, amino acids have been deleted(e.g., a truncated version of the protein, such as a peptide orfragment), inserted, inverted, substituted and/or derivatized (e.g., byglycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol). Preferred homologues of a PUFA PKSprotein or domain are described in detail below. It is noted thathomologues can include synthetically produced homologues, naturallyoccurring allelic variants of a given protein or domain, or homologoussequences from organisms other than the organism from which thereference sequence was derived.

In general, the biological activity or biological action of a protein ordomain refers to any function(s) exhibited or performed by the proteinor domain that is ascribed to the naturally occurring form of theprotein or domain as measured or observed in vivo (i.e., in the naturalphysiological environment of the protein) or in vitro (i.e., underlaboratory conditions). Biological activities of PUFA PKS systems andthe individual proteins/domains that make up a PUFA PKS system have beendescribed in detail elsewhere herein. Modifications of a protein ordomain, such as in a homologue or mimetic (discussed below), may resultin proteins or domains having the same biological activity as thenaturally occurring protein or domain, or in proteins or domains havingdecreased or increased biological activity as compared to the naturallyoccurring protein or domain. Modifications which result in a decrease inexpression or a decrease in the activity of the protein or domain, canbe referred to as inactivation (complete or partial), down-regulation,or decreased action of a protein or domain. Similarly, modificationswhich result in an increase in expression or an increase in the activityof the protein or domain, can be referred to as amplification,overproduction, activation, enhancement, up-regulation or increasedaction of a protein or domain. A functional domain of a PUFA PKS systemis a domain (i.e., a domain can be a portion of a protein) that iscapable of performing a biological function (i.e., has biologicalactivity).

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subject to human manipulation), itsnatural milieu being the genome or chromosome in which the nucleic acidmolecule is found in nature. As such, “isolated” does not necessarilyreflect the extent to which the nucleic acid molecule has been purified,but indicates that the molecule does not include an entire genome or anentire chromosome in which the nucleic acid molecule is found in nature.An isolated nucleic acid molecule can include a gene. An isolatednucleic acid molecule that includes a gene is not a fragment of achromosome that includes such gene, but rather includes the codingregion and regulatory regions associated with the gene, but noadditional genes naturally found on the same chromosome. An isolatednucleic acid molecule can also include a specified nucleic acid sequenceflanked by (i.e., at the 5′ and/or the 3′ end of the sequence)additional nucleic acids that do not normally flank the specifiednucleic acid sequence in nature (i.e., heterologous sequences). Isolatednucleic acid molecule can include DNA, RNA (e.g., mRNA), or derivativesof either DNA or RNA (e.g., cDNA). Although the phrase “nucleic acidmolecule” primarily refers to the physical nucleic acid molecule and thephrase “nucleic acid sequence” primarily refers to the sequence ofnucleotides on the nucleic acid molecule, the two phrases can be usedinterchangeably, especially with respect to a nucleic acid molecule, ora nucleic acid sequence, being capable of encoding a protein or domainof a protein.

Preferably, an isolated nucleic acid molecule of the present inventionis produced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning) or chemical synthesis. Isolatednucleic acid molecules include natural nucleic acid molecules andhomologues thereof, including, but not limited to, natural allelicvariants and modified nucleic acid molecules in which nucleotides havebeen inserted, deleted, substituted, and/or inverted in such a mannerthat such modifications provide the desired effect on PUFA PKS systembiological activity as described herein. Protein homologues (e.g.,proteins encoded by nucleic acid homologues) have been discussed indetail above.

A nucleic acid molecule homologue can be produced using a number ofmethods known to those skilled in the art (see, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LabsPress, 1989). For example, nucleic acid molecules can be modified usinga variety of techniques including, but not limited to, classicmutagenesis techniques and recombinant DNA techniques, such assite-directed mutagenesis, chemical treatment of a nucleic acid moleculeto induce mutations, restriction enzyme cleavage of a nucleic acidfragment, ligation of nucleic acid fragments, PCR amplification and/ormutagenesis of selected regions of a nucleic acid sequence, synthesis ofoligonucleotide mixtures and ligation of mixture groups to “build” amixture of nucleic acid molecules and combinations thereof. Nucleic acidmolecule homologues can be selected from a mixture of modified nucleicacids by screening for the function of the protein encoded by thenucleic acid and/or by hybridization with a wild-type gene.

The minimum size of a nucleic acid molecule of the present invention isa size sufficient to form a probe or oligonucleotide primer that iscapable of forming a stable hybrid (e.g., under moderate, high or veryhigh stringency conditions) with the complementary sequence of a nucleicacid molecule useful in the present invention, or of a size sufficientto encode an amino acid sequence having a biological activity of atleast one domain of a PUFA PKS system according to the presentinvention. As such, the size of the nucleic acid molecule encoding sucha protein can be dependent on nucleic acid composition and percenthomology or identity between the nucleic acid molecule and complementarysequence as well as upon hybridization conditions per se (e.g.,temperature, salt concentration, and formamide concentration). Theminimal size of a nucleic acid molecule that is used as anoligonucleotide primer or as a probe is typically at least about 12 toabout 15 nucleotides in length if the nucleic acid molecules are GC-richand at least about 15 to about 18 bases in length if they are AT-rich.There is no limit, other than a practical limit, on the maximal size ofa nucleic acid molecule of the present invention, in that the nucleicacid molecule can include a sequence sufficient to encode a biologicallyactive fragment of a domain of a PUFA PKS system, an entire domain of aPUFA PKS system, several domains within an open reading frame (Orf) of aPUFA PKS system, an entire Orf of a PUFA PKS system, or more than oneOrf of a PUFA PKS system.

In one embodiment of the present invention, an isolated nucleic acidmolecule comprises or consists essentially of a nucleic acid sequenceselected from the group of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, or biologically active fragments thereof. In one aspect, thenucleic acid sequence is selected from the group of: SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:17,SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27,SEQ ID NO:29, and SEQ ID NO:31. In one embodiment of the presentinvention, any of the above-described PUFA PKS amino acid sequences, aswell as homologues of such sequences, can be produced with from at leastone, and up to about 20, additional heterologous amino acids flankingeach of the C- and/or N-terminal end of the given amino acid sequence.The resulting protein or polypeptide can be referred to as “consistingessentially of” a given amino acid sequence. According to the presentinvention, the heterologous amino acids are a sequence of amino acidsthat are not naturally found (i.e., not found in nature, in vivo)flanking the given amino acid sequence or which would not be encoded bythe nucleotides that flank the naturally occurring nucleic acid sequenceencoding the given amino acid sequence as it occurs in the gene, if suchnucleotides in the naturally occurring sequence were translated usingstandard codon usage for the organism from which the given amino acidsequence is derived. Similarly, the phrase “consisting essentially of”,when used with reference to a nucleic acid sequence herein, refers to anucleic acid sequence encoding a given amino acid sequence that can beflanked by from at least one, and up to as many as about 60, additionalheterologous nucleotides at each of the 5′ and/or the 3′ end of thenucleic acid sequence encoding the given amino acid sequence. Theheterologous nucleotides are not naturally found (i.e., not found innature, in vivo) flanking the nucleic acid sequence encoding the givenamino acid sequence as it occurs in the natural gene.

The present invention also includes an isolated nucleic acid moleculecomprising a nucleic acid sequence encoding an amino acid sequencehaving a biological activity of at least one domain of a PUFA PKSsystem. In one aspect, such a nucleic acid sequence encodes a homologueof any of the Schizochytrium PUFA PKS ORFs or domains, including: SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13,SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26,SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32, wherein the homologue has abiological activity of at least one domain of a PUFA PKS system asdescribed previously herein.

In one aspect of the invention, a homologue of a Schizochytrium PUFA PKSprotein or domain encompassed by the present invention comprises anamino acid sequence that is at least about 60% identical to at least 500consecutive amino acids of an amino acid sequence chosen from: SEQ IDNO:2, SEQ ID NO:4, and SEQ ID NO:6; wherein said amino acid sequence hasa biological activity of at least one domain of a PUFA PKS system. In afurther aspect, the amino acid sequence of the homologue is at leastabout 60% identical to at least about 600 consecutive amino acids, andmore preferably to at least about 700 consecutive amino acids, and morepreferably to at least about 800 consecutive amino acids, and morepreferably to at least about 900 consecutive amino acids, and morepreferably to at least about 1000 consecutive amino acids, and morepreferably to at least about 1100 consecutive amino acids, and morepreferably to at least about 1200 consecutive amino acids, and morepreferably to at least about 1300 consecutive amino acids, and morepreferably to at least about 1400 consecutive amino acids, and morepreferably to at least about 1500 consecutive amino acids of any of SEQID NO:2, SEQ ID NO:4 and SEQ ID NO:6, or to the full length of SEQ IDNO:6. In a further aspect, the amino acid sequence of the homologue isat least about 60% identical to at least about 1600 consecutive aminoacids, and more preferably to at least about 1700 consecutive aminoacids, and more preferably to at least about 1800 consecutive aminoacids, and more preferably to at least about 1900 consecutive aminoacids, and more preferably to at least about 2000 consecutive aminoacids of any of SEQ ID NO:2 or SEQ ID NO:4, or to the full length of SEQID NO:4. In a further aspect, the amino acid sequence of the homologueis at least about 60% identical to at least about 2100 consecutive aminoacids, and more preferably to at least about 2200 consecutive aminoacids, and more preferably to at least about 2300 consecutive aminoacids, and more preferably to at least about 2400 consecutive aminoacids, and more preferably to at least about 2500 consecutive aminoacids, and more preferably to at least about 2600 consecutive aminoacids, and more preferably to at least about 2700 consecutive aminoacids, and more preferably to at least about 2800 consecutive aminoacids, and even more preferably, to the full length of SEQ ID NO:2.

In another aspect, a homologue of a Schizochytrium PUFA PKS protein ordomain encompassed by the present invention comprises an amino acidsequence that is at least about 65% identical, and more preferably atleast about 70% identical, and more preferably at least about 75%identical, and more preferably at least about 80% identical, and morepreferably at least about 85% identical, and more preferably at leastabout 90% identical, and more preferably at least about 95% identical,and more preferably at least about 96% identical, and more preferably atleast about 97% identical, and more preferably at least about 98%identical, and more preferably at least about 99% identical to an aminoacid sequence chosen from: SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6,over any of the consecutive amino acid lengths described in theparagraph above, wherein the amino acid sequence has a biologicalactivity of at least one domain of a PUFA PKS system.

In one aspect of the invention, a homologue of a Schizochytrium PUFA PKSprotein or domain encompassed by the present invention comprises anamino acid sequence that is at least about 60% identical to an aminoacid sequence chosen from: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, or SEQ ID NO:32, wherein said amino acid sequencehas a biological activity of at least one domain of a PUFA PKS system.In a further aspect, the amino acid sequence of the homologue is atleast about 65% identical, and more preferably at least about 70%identical, and more preferably at least about 75% identical, and morepreferably at least about 80% identical, and more preferably at leastabout 85% identical, and more preferably at least about 90% identical,and more preferably at least about 95% identical, and more preferably atleast about 96% identical, and more preferably at least about 97%identical, and more preferably at least about 98% identical, and morepreferably at least about 99% identical to an amino acid sequence chosenfrom: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, wherein the amino acid sequence has a biologicalactivity of at least one domain of a PUFA PKS system.

According to the present invention, the term “contiguous” or“consecutive”, with regard to nucleic acid or amino acid sequencesdescribed herein, means to be connected in an unbroken sequence. Forexample, for a first sequence to comprise 30 contiguous (or consecutive)amino acids of a second sequence, means that the first sequence includesan unbroken sequence of 30 amino acid residues that is 100% identical toan unbroken sequence of 30 amino acid residues in the second sequence.Similarly, for a first sequence to have “100% identity” with a secondsequence means that the first sequence exactly matches the secondsequence with no gaps between nucleotides or amino acids.

As used herein, unless otherwise specified, reference to a percent (%)identity refers to an evaluation of homology which is performed using:(1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acidsearches, blastn for nucleic acid searches, and blastX for nucleic acidsearches and searches of translated amino acids in all 6 open readingframes, all with standard default parameters, wherein the query sequenceis filtered for low complexity regions by default (described inAltschul, S. F., Madden, T. L., Schääffer, A. A., Zhang, J., Zhang, Z.,Miller, W. & Lipman, D. J. (1997) “Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs.” Nucleic Acids Res.25:3389-3402, incorporated herein by reference in its entirety); (2) aBLAST 2 alignment (using the parameters described below); (3) and/orPSI-BLAST with the standard default parameters (Position-SpecificIterated BLAST). It is noted that due to some differences in thestandard parameters between BLAST 2.0 Basic BLAST and BLAST 2, twospecific sequences might be recognized as having significant homologyusing the BLAST 2 program, whereas a search performed in BLAST 2.0 BasicBLAST using one of the sequences as the query sequence may not identifythe second sequence in the top matches. In addition, PSI-BLAST providesan automated, easy-to-use version of a “profile” search, which is asensitive way to look for sequence homologues. The program firstperforms a gapped BLAST database search. The PSI-BLAST program uses theinformation from any significant alignments returned to construct aposition-specific score matrix, which replaces the query sequence forthe next round of database searching. Therefore, it is to be understoodthat percent identity can be determined by using any one of theseprograms.

Two specific sequences can be aligned to one another using BLAST 2sequence as described in Tatusova and Madden, (1999), “Blast 2sequences—a new tool for comparing protein and nucleotide sequences”,FEMS Microbiol Lett. 174:247-250, incorporated herein by reference inits entirety. BLAST 2 sequence alignment is performed in blastp orblastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search(BLAST 2.0) between the two sequences allowing for the introduction ofgaps (deletions and insertions) in the resulting alignment. For purposesof clarity herein, a BLAST 2 sequence alignment is performed using thestandard default parameters as follows.

For blastn, using 0 BLOSUM62 matrix:

Reward for match=1

Penalty for mismatch=−2

Open gap (5) and extension gap (2) penalties

gap x_dropoff (50) expect (10) word size (11) filter (on)

For blastp, using 0 BLOSUM62 matrix:

Open gap (11) and extension gap (1) penalties

gap x_dropoff (50) expect (10) word size (3) filter (on).

In another embodiment of the invention, an amino acid sequence havingthe biological activity of at least one domain of a PUFA PKS system ofthe present invention includes an amino acid sequence that issufficiently similar to a naturally occurring PUFA PKS protein orpolypeptide that a nucleic acid sequence encoding the amino acidsequence is capable of hybridizing under moderate, high, or very highstringency conditions (described below) to (i.e., with) a nucleic acidmolecule encoding the naturally occurring PUFA PKS protein orpolypeptide (i.e., to the complement of the nucleic acid strand encodingthe naturally occurring PUFA PKS protein or polypeptide). Preferably, anamino acid sequence having the biological activity of at least onedomain of a PUFA PKS system of the present invention is encoded by anucleic acid sequence that hybridizes under moderate, high or very highstringency conditions to the complement of a nucleic acid sequence thatencodes a protein comprising an amino acid sequence represented by anyof SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:13, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32. Methods to deduce acomplementary sequence are known to those skilled in the art. It shouldbe noted that since amino acid sequencing and nucleic acid sequencingtechnologies are not entirely error-free, the sequences presentedherein, at best, represent apparent sequences of PUFA PKS domains andproteins of the present invention.

As used herein, hybridization conditions refer to standard hybridizationconditions under which nucleic acid molecules are used to identifysimilar nucleic acid molecules. Such standard conditions are disclosed,for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Labs Press, 1989. Sambrook et al., ibid., isincorporated by reference herein in its entirety (see specifically,pages 9.31-9.62). In addition, formulae to calculate the appropriatehybridization and wash conditions to achieve hybridization permittingvarying degrees of mismatch of nucleotides are disclosed, for example,in Meinkoth et al., 1984, Anal Biochem. 138, 267-284; Meinkoth et al.,ibid., is incorporated by reference herein in its entirety.

More particularly, moderate stringency hybridization and washingconditions, as referred to herein, refer to conditions which permitisolation of nucleic acid molecules having at least about 70% nucleicacid sequence identity with the nucleic acid molecule being used toprobe in the hybridization reaction (i.e., conditions permitting about30% or less mismatch of nucleotides). High stringency hybridization andwashing conditions, as referred to herein, refer to conditions whichpermit isolation of nucleic acid molecules having at least about 80%nucleic acid sequence identity with the nucleic acid molecule being usedto probe in the hybridization reaction (i.e., conditions permittingabout 20% or less mismatch of nucleotides). Very high stringencyhybridization and washing conditions, as referred to herein, refer toconditions which permit isolation of nucleic acid molecules having atleast about 90% nucleic acid sequence identity with the nucleic acidmolecule being used to probe in the hybridization reaction (i.e.,conditions permitting about 10% or less mismatch of nucleotides). Asdiscussed above, one of skill in the art can use the formulae inMeinkoth et al., ibid. to calculate the appropriate hybridization andwash conditions to achieve these particular levels of nucleotidemismatch. Such conditions will vary, depending on whether DNA:RNA orDNA:DNA hybrids are being formed. Calculated melting temperatures forDNA:DNA hybrids are 10° C. less than for DNA:RNA hybrids. In particularembodiments, stringent hybridization conditions for DNA:DNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 20° C. and about 35° C. (lower stringency),more preferably, between about 28° C. and about 40° C. (more stringent),and even more preferably, between about 35° C. and about 45° C. (evenmore stringent), with appropriate wash conditions. In particularembodiments, stringent hybridization conditions for DNA:RNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 30° C. and about 45° C., more preferably,between about 38° C. and about 50° C., and even more preferably, betweenabout 45° C. and about 55° C., with similarly stringent wash conditions.These values are based on calculations of a melting temperature formolecules larger than about 100 nucleotides, 0% formamide and a G+Ccontent of about 40%. Alternatively, T_(m) can be calculated empiricallyas set forth in Sambrook et al., supra, pages 9.31 to 9.62. In general,the wash conditions should be as stringent as possible, and should beappropriate for the chosen hybridization conditions. For example,hybridization conditions can include a combination of salt andtemperature conditions that are approximately 20-25° C. below thecalculated T_(m) of a particular hybrid, and wash conditions typicallyinclude a combination of salt and temperature conditions that areapproximately 12-20° C. below the calculated T_(m) of the particularhybrid. One example of hybridization conditions suitable for use withDNA:DNA hybrids includes a 2-24 hour hybridization in 6×SSC (50%formamide) at about 42° C., followed by washing steps that include oneor more washes at room temperature in about 2×SSC, followed byadditional washes at higher temperatures and lower ionic strength (e.g.,at least one wash as about 37° C. in about 0.1×-0.5×SSC, followed by atleast one wash at about 68° C. in about 0.1×-0.5×SSC).

Another embodiment of the present invention includes a recombinantnucleic acid molecule comprising a recombinant vector and a nucleic acidmolecule comprising a nucleic acid sequence encoding an amino acidsequence having a biological activity of at least one domain of a PUFAPKS system as described herein. Such nucleic acid sequences aredescribed in detail above. According to the present invention, arecombinant vector is an engineered (i.e., artificially produced)nucleic acid molecule that is used as a tool for manipulating a nucleicacid sequence of choice and for introducing such a nucleic acid sequenceinto a host cell. The recombinant vector is therefore suitable for usein cloning, sequencing, and/or otherwise manipulating the nucleic acidsequence of choice, such as by expressing and/or delivering the nucleicacid sequence of choice into a host cell to form a recombinant cell.Such a vector typically contains heterologous nucleic acid sequences,that is nucleic acid sequences that are not naturally found adjacent tonucleic acid sequence to be cloned or delivered, although the vector canalso contain regulatory nucleic acid sequences (e.g., promoters,untranslated regions) which are naturally found adjacent to nucleic acidmolecules of the present invention or which are useful for expression ofthe nucleic acid molecules of the present invention (discussed in detailbelow). The vector can be either RNA or DNA, either prokaryotic oreukaryotic, and typically is a plasmid. The vector can be maintained asan extrachromosomal element (e.g., a plasmid) or it can be integratedinto the chromosome of a recombinant organism (e.g., a microbe or aplant). The entire vector can remain in place within a host cell, orunder certain conditions, the plasmid DNA can be deleted, leaving behindthe nucleic acid molecule of the present invention. The integratednucleic acid molecule can be under chromosomal promoter control, undernative or plasmid promoter control, or under a combination of severalpromoter controls. Single or multiple copies of the nucleic acidmolecule can be integrated into the chromosome. A recombinant vector ofthe present invention can contain at least one selectable marker.

In one embodiment, a recombinant vector used in a recombinant nucleicacid molecule of the present invention is an expression vector. As usedherein, the phrase “expression vector” is used to refer to a vector thatis suitable for production of an encoded product (e.g., a protein ofinterest). In this embodiment, a nucleic acid sequence encoding theproduct to be produced (e.g., a PUFA PKS domain) is inserted into therecombinant vector to produce a recombinant nucleic acid molecule. Thenucleic acid sequence encoding the protein to be produced is insertedinto the vector in a manner that operatively links the nucleic acidsequence to regulatory sequences in the vector which enable thetranscription and translation of the nucleic acid sequence within therecombinant host cell.

In another embodiment, a recombinant vector used in a recombinantnucleic acid molecule of the present invention is a targeting vector. Asused herein, the phrase “targeting vector” is used to refer to a vectorthat is used to deliver a particular nucleic acid molecule into arecombinant host cell, wherein the nucleic acid molecule is used todelete or inactivate an endogenous gene within the host cell ormicroorganism (i.e., used for targeted gene disruption or knock-outtechnology). Such a vector may also be known in the art as a “knock-out”vector. In one aspect of this embodiment, a portion of the vector, butmore typically, the nucleic acid molecule inserted into the vector(i.e., the insert), has a nucleic acid sequence that is homologous to anucleic acid sequence of a target gene in the host cell (i.e., a genewhich is targeted to be deleted or inactivated). The nucleic acidsequence of the vector insert is designed to bind to the target genesuch that the target gene and the insert undergo homologousrecombination, whereby the endogenous target gene is deleted,inactivated or attenuated (i.e., by at least a portion of the endogenoustarget gene being mutated or deleted).

Typically, a recombinant nucleic acid molecule includes at least onenucleic acid molecule of the present invention operatively linked to oneor more transcription control sequences. As used herein, the phrase“recombinant molecule” or “recombinant nucleic acid molecule” primarilyrefers to a nucleic acid molecule or nucleic acid sequence operativelylinked to a transcription control sequence, but can be usedinterchangeably with the phrase “nucleic acid molecule”, when suchnucleic acid molecule is a recombinant molecule as discussed herein.According to the present invention, the phrase “operatively linked”refers to linking a nucleic acid molecule to a transcription controlsequence in a manner such that the molecule is able to be expressed whentransfected (i.e., transformed, transduced, transfected, conjugated orconduced) into a host cell. Transcription control sequences aresequences which control the initiation, elongation, or termination oftranscription. Particularly important transcription control sequencesare those which control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in a host cell or organism into which the recombinant nucleicacid molecule is to be introduced.

Recombinant nucleic acid molecules of the present invention can alsocontain additional regulatory sequences, such as translation regulatorysequences, origins of replication, and other regulatory sequences thatare compatible with the recombinant cell. In one embodiment, arecombinant molecule of the present invention, including those which areintegrated into the host cell chromosome, also contains secretorysignals (i.e., signal segment nucleic acid sequences) to enable anexpressed protein to be secreted from the cell that produces theprotein. Suitable signal segments include a signal segment that isnaturally associated with the protein to be expressed or anyheterologous signal segment capable of directing the secretion of theprotein according to the present invention. In another embodiment, arecombinant molecule of the present invention comprises a leadersequence to enable an expressed protein to be delivered to and insertedinto the membrane of a host cell. Suitable leader sequences include aleader sequence that is naturally associated with the protein, or anyheterologous leader sequence capable of directing the delivery andinsertion of the protein to the membrane of a cell.

The present inventors have found that the Schizochytrium PUFA PKS Orfs Aand B are closely linked in the genome and region between the Orfs hasbeen sequenced. The Orfs are oriented in opposite directions and 4244base pairs separate the start (ATG) codons (i.e. they are arranged asfollows: 3′OrfA5′-4244 bp-5′OrfB3′). Examination of the 4244 bpintergenic region did not reveal any obvious Orfs (no significantmatches were found on a BlastX search). Both Orfs A and B are highlyexpressed in Schizochytrium, at least during the time of oil production,implying that active promoter elements are embedded in this intergenicregion. These genetic elements are believed to have utility as abi-directional promoter sequence for transgenic applications. Forexample, in a preferred embodiment, one could clone this region, placeany genes of interest at each end and introduce the construct intoSchizochytrium (or some other host in which the promoters can be shownto function). It is predicted that the regulatory elements, under theappropriate conditions, would provide for coordinated, high levelexpression of the two introduced genes. The complete nucleotide sequencefor the regulatory region containing Schizochytrium PUFA PKS regulatoryelements (e.g., a promoter) is represented herein as SEQ ID NO:36.

In a similar manner, OrfC is highly expressed in Schizochytrium duringthe time of oil production and regulatory elements are expected toreside in the region upstream of its start codon. A region of genomicDNA upstream of OrfC has been cloned and sequenced and is representedherein as (SEQ ID NO:37). This sequence contains the 3886 nt immediatelyupstream of the OrfC start codon. Examination of this region did notreveal any obvious Orfs (i.e., no significant matches were found on aBlastX search). It is believed that regulatory elements contained inthis region, under the appropriate conditions, will provide forhigh-level expression of a gene placed behind them. Additionally, underthe appropriate conditions, the level of expression may be coordinatedwith genes under control of the A-B intergenic region (SEQ ID NO:36).

Therefore, in one embodiment, a recombinant nucleic acid molecule usefulin the present invention, as disclosed herein, can include a PUFA PKSregulatory region contained within SEQ ID NO:36 and/or SEQ ID NO:37.Such a regulatory region can include any portion (fragment) of SEQ IDNO:36 and/or SEQ ID NO:37 that has at least basal PUFA PKStranscriptional activity.

One or more recombinant molecules of the present invention can be usedto produce an encoded product (e.g., a PUFA PKS domain, protein, orsystem) of the present invention. In one embodiment, an encoded productis produced by expressing a nucleic acid molecule as described hereinunder conditions effective to produce the protein. A preferred method toproduce an encoded protein is by transfecting a host cell with one ormore recombinant molecules to form a recombinant cell. Suitable hostcells to transfect include, but are not limited to, any bacterial,fungal (e.g., yeast), insect, plant or animal cell that can betransfected. Host cells can be either untransfected cells or cells thatare already transfected with at least one other recombinant nucleic acidmolecule.

According to the present invention, the term “transfection” is used torefer to any method by which an exogenous nucleic acid molecule (i.e., arecombinant nucleic acid molecule) can be inserted into a cell. The term“transformation” can be used interchangeably with the term“transfection” when such term is used to refer to the introduction ofnucleic acid molecules into microbial cells, such as algae, bacteria andyeast. In microbial systems, the term “transformation” is used todescribe an inherited change due to the acquisition of exogenous nucleicacids by the microorganism and is essentially synonymous with the term“transfection.” However, in animal cells, transformation has acquired asecond meaning which can refer to changes in the growth properties ofcells in culture after they become cancerous, for example. Therefore, toavoid confusion, the term “transfection” is preferably used with regardto the introduction of exogenous nucleic acids into animal cells, andthe term “transfection” will be used herein to generally encompasstransfection of animal cells, plant cells and transformation ofmicrobial cells, to the extent that the terms pertain to theintroduction of exogenous nucleic acids into a cell. Therefore,transfection techniques include, but are not limited to, transformation,particle bombardment, electroporation, microinjection, lipofection,adsorption, infection and protoplast fusion.

It will be appreciated by one skilled in the art that use of recombinantDNA technologies can improve control of expression of transfectednucleic acid molecules by manipulating, for example, the number ofcopies of the nucleic acid molecules within the host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Additionally, thepromoter sequence might be genetically engineered to improve the levelof expression as compared to the native promoter. Recombinant techniquesuseful for controlling the expression of nucleic acid molecules include,but are not limited to, integration of the nucleic acid molecules intoone or more host cell chromosomes, addition of vector stabilitysequences to plasmids, substitutions or modifications of transcriptioncontrol signals (e.g., promoters, operators, enhancers), substitutionsor modifications of translational control signals (e.g., ribosomebinding sites, Shine-Dalgarno sequences), modification of nucleic acidmolecules to correspond to the codon usage of the host cell, anddeletion of sequences that destabilize transcripts.

General discussion above with regard to recombinant nucleic acidmolecules and transfection of host cells is intended to be applied toany recombinant nucleic acid molecule discussed herein, including thoseencoding any amino acid sequence having a biological activity of atleast one domain from a PUFA PKS, those encoding amino acid sequencesfrom other PKS systems, and those encoding other proteins or domains.

This invention also relates to the use of a novel method to identify amicroorganism that has a PUFA PKS system that is homologous instructure, domain organization and/or function to a Schizochytrium PUFAPKS system. In one embodiment, the microorganism is a non-bacterialmicroorganism, and preferably, the microorganism identified by thismethod is a eukaryotic microorganism. In addition, this inventionrelates to the microorganisms identified by such method and to the useof these microorganisms and the PUFA PKS systems from thesemicroorganisms in the various applications for a PUFA PKS system (e.g.,genetically modified organisms and methods of producing bioactivemolecules) according to the present invention. The unique screeningmethod described and demonstrated herein enables the rapididentification of new microbial strains containing a PUFA PKS systemhomologous to the Schizochytrium PUFA PKS system of the presentinvention. Applicants have used this method to discover and discloseherein that a Thraustochytrium microorganism contains a PUFA PKS systemthat is homologous to that found in Schizochytrium. This discovery isdescribed in detail in Example 2 below.

Microbial organisms with a PUFA PKS system similar to that found inSchizochytrium, such as the Thraustochytrium microorganism discovered bythe present inventors and described in Example 2, can be readilyidentified/isolated/screened by the following methods used separately orin any combination of these methods.

In general, the method to identify a non-bacterial microorganism thathas a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systemincludes a first step of (a) selecting a microorganism that produces atleast one PUFA; and a second step of (b) identifying a microorganismfrom (a) that has an ability to produce increased PUFAs under dissolvedoxygen conditions of less than about 5% of saturation in thefermentation medium, as compared to production of PUFAs by saidmicroorganism under dissolved oxygen conditions of greater than 5% ofsaturation, more preferably 10% of saturation, more preferably greaterthan 15% of saturation and more preferably greater than 20% ofsaturation in the fermentation medium. A microorganism that produces atleast one PUFA and has an ability to produce increased PUFAs underdissolved oxygen conditions of less than about 5% of saturation isidentified as a candidate for containing a PUFA PKS system. Subsequentto identifying a microorganism that is a strong candidate for containinga PUFA PKS system, the method can include an additional step (c) ofdetecting whether the organism identified in step (b) comprises a PUFAPKS system.

In one embodiment of the present invention, step (b) is performed byculturing the microorganism selected for the screening process in lowoxygen/anoxic conditions and aerobic conditions, and, in addition tomeasuring PUFA content in the organism, the fatty acid profile isdetermined, as well as fat content. By comparing the results under lowoxygen/anoxic conditions with the results under aerobic conditions, themethod provides a strong indication of whether the test microorganismcontains a PUFA PKS system of the present invention. This preferredembodiment is described in detail below.

Initially, microbial strains to be examined for the presence of a PUFAPKS system are cultured under aerobic conditions to induce production ofa large number of cells (microbial biomass). As one element of theidentification process, these cells are then placed under low oxygen oranoxic culture conditions (e.g., dissolved oxygen less than about 5% ofsaturation, more preferably less than about 2%, even more preferablyless than about 1%, and most preferably dissolved oxygen of about 0% ofsaturation in the culture medium) and allowed to grow for approximatelyanother 24-72 hours. In this process, the microorganisms should becultured at a temperature greater than about 15° C., and more preferablygreater than about 20° C., and even more preferably greater than about25° C., and even more preferably greater than 30° C. The low or anoxicculture environment can be easily maintained in culture chambers capableof inducing this type of atmospheric environment in the chamber (andthus in the cultures) or by culturing the cells in a manner that inducesthe low oxygen environment directly in the culture flask/vessel itself.

In a preferred culturing method, the microbes can be cultured in shakeflasks which, instead of normally containing a small amount of culturemedium—less than about 50% of total capacity and usually less than about25% of total capacity—to keep the medium aerated as it is shaken on ashaker table, are instead filled to greater than about 50% of theircapacity, and more preferably greater than about 60%, and mostpreferably greater than about 75% of their capacity with culture medium.High loading of the shake flask with culture medium prevents it frommixing very well in the flask when it is placed on a shaker table,preventing oxygen diffusion into the culture. Therefore as the microbesgrow, they use up the existing oxygen in the medium and naturally createa low or no oxygen environment in the shake flask.

After the culture period, the cells are harvested and analyzed forcontent of bioactive compounds of interest (e.g., lipids), but mostparticularly, for compounds containing two or more unsaturated bonds,and more preferably three or more double bonds, and even more preferablyfour or more double bonds. For lipids, those strains possessing suchcompounds at greater than about 5%, and more preferably greater thanabout 10%, and more preferably greater than about 15%, and even morepreferably greater than about 20% of the dry weight of the microorganismare identified as predictably containing a novel PKS system of the typedescribed above. For other bioactive compounds, such as antibiotics orcompounds that are synthesized in smaller amounts, those strainspossessing such compounds at greater than about 0.5%, and morepreferably greater than about 0.1%, and more preferably greater thanabout 0.25%, and more preferably greater than about 0.5%, and morepreferably greater than about 0.75%, and more preferably greater thanabout 1%, and more preferably greater than about 2.5%, and morepreferably greater than about 5% of the dry weight of the microorganismare identified as predictably containing a novel PKS system of the typedescribed above.

Alternatively, or in conjunction with this method, prospective microbialstrains containing novel PUFA PKS systems as described herein can beidentified by examining the fatty acid profile of the strain (obtainedby culturing the organism or through published or other readilyavailable sources). If the microbe contains greater than about 30%, andmore preferably greater than about 40%, and more preferably greater thanabout 45%, and even more preferably greater than about 50% of its totalfatty acids as C14:0, C16:0 and/or C16:1, while also producing at leastone long chain fatty acid with three or more unsaturated bonds, and morepreferably 4 or more double bonds, and more preferably 5 or more doublebonds, and even more preferably 6 or more double bonds, then thismicrobial strain is identified as a likely candidate to possess a novelPUFA PKS system of the type described in this invention. Screening thisorganism under the low oxygen conditions described above, and confirmingproduction of bioactive molecules containing two or more unsaturatedbonds would suggest the existence of a novel PUFA PKS system in theorganism, which could be further confirmed by analysis of the microbes'genome.

The success of this method can also be enhanced by screening eukaryoticstrains that are known to contain C17:0 and or C17:1 fatty acids (inconjunction with the large percentages of C14:0, C16:0 and C16:1 fattyacids described above)—because the C17:0 and C17:1 fatty acids arepotential markers for a bacterial (prokaryotic) based or influencedfatty acid production system. Another marker for identifying strainscontaining novel PUFA PKS systems is the production of simple fatty acidprofiles by the organism. According to the present invention, a “simplefatty acid profile” is defined as 8 or fewer fatty acids being producedby the strain at levels greater than 10% of total fatty acids.

Use of any of these methods or markers (singly or preferably incombination) would enable one of skill in the art to readily identifymicrobial strains that are highly predicted to contain a novel PUFA PKSsystem of the type described in this invention.

In a preferred embodiment combining many of the methods and markersdescribed above, a novel biorational screen (using shake flask cultures)has been developed for detecting microorganisms containing PUFAproducing PKS systems. This screening system is conducted as follows:

A portion of a culture of the strain/microorganism to be tested isplaced in 250 mL baffled shake flask with 50 mL culture media (aerobictreatment), and another portion of culture of the same strain is placedin a 250 mL non-baffled shake flask with 200 mL culture medium(anoxic/low oxygen treatment). Various culture media can be employeddepending on the type and strain of microorganism being evaluated. Bothflasks are placed on a shaker table at 200 rpm. After 48-72 hr ofculture time, the cultures are harvested by centrifugation and the cellsare analyzed for fatty acid methyl ester content via gas chromatographyto determine the following data for each culture: (1) fatty acidprofile; (2) PUFA content; and (3) fat content (approximated as amounttotal fatty acids/cell dry weight).

These data are then analyzed asking the following five questions(Yes/No):

Comparing the data from the low O₂/anoxic flask with the data from theaerobic flask:

(1) Did the DHA (or other PUFA content) (as % FAME (fatty acid methylesters)) stay about the same or preferably increased in the low oxygenculture compared to the aerobic culture?

(2) Is C14:0+C16:0+C16:1 greater than about 40% TFA in the anoxicculture?

(3) Are there very little (<1% as FAME) or no precursors(C18:3n-3+C18:2n-6+C18:3n-6) to the conventional oxygen dependentelongase/desaturase pathway in the anoxic culture?

(4) Did fat content (as amount total fatty acids/cell dry weight)increase in the low oxygen culture compared to the aerobic culture?

(5) Did DHA (or other PUFA content) increase as % cell dry weight in thelow oxygen culture compared to the aerobic culture?

If the first three questions are answered yes, this is a good indicationthat the strain contains a PKS genetic system for making long chainPUFAs. The more questions that are answered yes (preferably the firstthree questions must be answered yes), the stronger the indication thatthe strain contains such a PKS genetic system. If all five questions areanswered yes, then there is a very strong indication that the straincontains a PKS genetic system for making long chain PUFAs. The lack of18:3n-3/18:2n-6/18:3n-6 would indicate that the low oxygen conditionswould have turned off or inhibited the conventional pathway for PUFAsynthesis. A high 14:0/16:0/16:1 fatty is an preliminary indicator of abacterially influenced fatty acid synthesis profile (the presence ofC17:0 and 17:1 is also and indicator of this) and of a simple fatty acidprofile. The increased PUFA synthesis and PUFA containing fat synthesisunder the low oxygen conditions is directly indicative of a PUFA PKSsystem, since this system does not require oxygen to make highlyunsaturated fatty acids.

Finally, in the identification method of the present invention, once astrong candidate is identified, the microbe is preferably screened todetect whether or not the microbe contains a PUFA PKS system. Forexample, the genome of the microbe can be screened to detect thepresence of one or more nucleic acid sequences that encode a domain of aPUFA PKS system as described herein. Preferably, this step of detectionincludes a suitable nucleic acid detection method, such ashybridization, amplification and or sequencing of one or more nucleicacid sequences in the microbe of interest. The probes and/or primersused in the detection methods can be derived from any known PUFA PKSsystem, including the marine bacteria PUFA PKS systems described in U.S.Pat. No. 6,140,486, or the Thraustochytrid PUFA PKS systems described inU.S. application Ser. No. 09/231,899 and herein. Once novel PUFA PKSsystems are identified, the genetic material from these systems can alsobe used to detect additional novel PUFA PKS systems. Methods ofhybridization, amplification and sequencing of nucleic acids for thepurpose of identification and detection of a sequence are well known inthe art. Using these detection methods, sequence homology and domainstructure (e.g., the presence, number and/or arrangement of various PUFAPKS functional domains) can be evaluated and compared to the known PUFAPKS systems described herein.

In some embodiments, a PUFA PKS system can be identified usingbiological assays. For example, in U.S. application Ser. No. 09/231,899,Example 7, the results of a key experiment using a well-known inhibitorof some types of fatty acid synthesis systems, i.e., thiolactomycin, isdescribed. The inventors showed that the synthesis of PUFAs in wholecells of Schizochytrium could be specifically blocked without blockingthe synthesis of short chain saturated fatty acids. The significance ofthis result is as follows: the inventors knew from analysis of cDNAsequences from Schizochytrium that a Type I fatty acid synthase systemis present in Schizochytrium. It was known that thiolactomycin does notinhibit Type I FAS systems, and this is consistent with the inventors'data—i.e., production of the saturated fatty acids (primarily C14:0 andC16:0 in Schizochytrium) was not inhibited by the thiolactomycintreatment. There are no indications in the literature or in theinventors' own data that thiolactomycin has any inhibitory effect on theelongation of C14:0 or C16:0 fatty acids or their desaturation (i.e. theconversion of short chain saturated fatty acids to PUFAs by theclassical pathway). Therefore, the fact that the PUFA production inSchizochytrium was blocked by thiolactomycin strongly indicates that theclassical PUFA synthesis pathway does not produce the PUFAs inSchizochytrium, but rather that a different pathway of synthesis isinvolved. Further, it had previously been determined that the ShewanellaPUFA PKS system is inhibited by thiolactomycin (note that the PUFA PKSsystem of the present invention has elements of both Type I and Type IIsystems), and it was known that thiolactomycin is an inhibitor of TypeII FAS systems (such as that found in E. coli). Therefore, thisexperiment indicated that Schizochytrium produced PUFAs as a result of apathway not involving the Type I FAS. A similar rationale and detectionstep could be used to detect a PUFA PKS system in a microbe identifiedusing the novel screening method disclosed herein.

In addition, Example 3 shows additional biochemical data which providesevidence that PUFAs in Schizochytrium are not produced by the classicalpathway (i.e., precursor product kinetics between C16:0 and DHA are notobserved in whole cells and, in vitro PUFA synthesis can be separatedfrom the membrane fraction—all of the fatty acid desaturases of theclassical PUFA synthesis pathway, with the exception of the delta 9desaturase which inserts the first double bond of the series, areassociated with cellular membranes). This type of biochemical data couldbe used to detect PUFA PKS activity in microbe identified by the novelscreening method described above.

Preferred microbial strains to screen using the screening/identificationmethod of the present invention are chosen from the group consisting of:bacteria, algae, fungi, protozoa or protists, but most preferably fromthe eukaryotic microbes consisting of algae, fungi, protozoa andprotists. These microbes are preferably capable of growth and productionof the bioactive compounds containing two or more unsaturated bonds attemperatures greater than about 15° C., more preferably greater thanabout 20° C., even more preferably greater than about 25° C. and mostpreferably greater than about 30° C.

In some embodiments of this method of the present invention, novelbacterial PUFA PKS systems can be identified in bacteria that producePUFAs at temperatures exceeding about 20° C., preferably exceeding about25° C. and even more preferably exceeding about 30° C. As describedpreviously herein, the marine bacteria, Shewanella and Vibrio marinus,described in U.S. Pat. No. 6,140,486, do not produce PUFAs at highertemperatures, which limits the usefulness of PUFA PKS systems derivedfrom these bacteria, particularly in plant applications under fieldconditions. Therefore, in one embodiment, the screening method of thepresent invention can be used to identify bacteria that have a PUFA PKSsystem which are capable of growth and PUFA production at highertemperatures (e.g., above about 20, 25, or 30° C.). In this embodiment,inhibitors of eukaryotic growth such as nystatin (antifungal) orcycloheximide (inhibitor of eukaryotic protein synthesis) can be addedto agar plates used to culture/select initial strains from watersamples/soil samples collected from the types of habitats/nichesdescribed below. This process would help select for enrichment ofbacterial strains without (or minimal) contamination of eukaryoticstrains. This selection process, in combination with culturing theplates at elevated temperatures (e.g. 30° C.), and then selectingstrains that produce at least one PUFA would initially identifycandidate bacterial strains with a PUFA PKS system that is operative atelevated temperatures (as opposed to those bacterial strains in theprior art which only exhibit PUFA production at temperatures less thanabout 20° C. and more preferably below about 5° C.).

Locations for collection of the preferred types of microbes forscreening for a PUFA PKS system according to the present inventioninclude any of the following: low oxygen environments (or locations nearthese types of low oxygen environments including in the guts of animalsincluding invertebrates that consume microbes or microbe-containingfoods (including types of filter feeding organisms), low or non-oxygencontaining aquatic habitats (including freshwater, saline and marine),and especially at- or near-low oxygen environments (regions) in theoceans. The microbial strains would preferably not be obligate anaerobesbut be adapted to live in both aerobic and low or anoxic environments.Soil environments containing both aerobic and low oxygen or anoxicenvironments would also excellent environments to find these organismsin and especially in these types of soil in aquatic habitats ortemporary aquatic habitats.

A particularly preferred microbial strain would be a strain (selectedfrom the group consisting of algae, fungi (including yeast), protozoa orprotists) that, during a portion of its life cycle, is capable ofconsuming whole bacterial cells (bacterivory) by mechanisms such asphagocytosis, phagotrophic or endocytic capability and/or has a stage ofits life cycle in which it exists as an amoeboid stage or nakedprotoplast. This method of nutrition would greatly increase thepotential for transfer of a bacterial PKS system into a eukaryotic cellif a mistake occurred and the bacterial cell (or its DNA) did not getdigested and instead are functionally incorporated into the eukaryoticcell.

Strains of microbes (other than the members of the Thraustochytrids)capable of bacterivory (especially by phagocytosis or endocytosis) canbe found in the following microbial classes (including but not limitedto example genera):

In the algae and algae-like microbes (including stramenopiles): of theclass Euglenophyceae (for example genera Euglena, and Peranema), theclass Chrysophyceae (for example the genus Ochromonas), the classDinobryaceae (for example the genera Dinobryon, Platychrysis, andChrysochromulina), the Dinophyceae (including the generaCrypthecodinium, Gymnodinium, Peridinium, Ceratium, Gyrodinium, andOxyrrhis), the class Cryptophyceae (for example the genera Cryptomonas,and Rhodomonas), the class Xanthophyceae (for example the genusOlisthodiscus) (and including forms of algae in which an amoeboid stageoccurs as in the flagellates Rhizochloridaceae, and zoospores/gametes ofAphanochaete pascheri, Bumilleria stigeoclonium and Vaucheria geminata),the class Eustigmatophyceae, and the class Prymnesiopyceae (includingthe genera Prymnesium and Diacronema).

In the Stramenopiles including the: Proteromonads, Opalines,Developayella, Diplophorys, Larbrinthulids, Thraustochytrids,Bicosecids, Oomycetes, Hypochytridiomycetes, Commation, Reticulosphaera,Pelagomonas, Pelapococcus, Ollicola, Aureococcus, Parmales,Raphidiophytes, Synurids, Rhizochromulinaales, Pedinellales,Dictyochales, Chrysomeridales, Sarcinochrysidales, Hydrurales,Hibberdiales, and Chromulinales.

In the Fungi: Class Myxomycetes (form myxamoebae)—slime molds, classAcrasieae including the orders Acrasiceae (for example the genusSappinia), class Guttulinaceae (for example the genera Guttulinopsis,and Guttulina), class Dictysteliaceae (for example the genera Acrasis,Dictyostelium, Polysphondylium, and Coenonia), and class Phycomyceaeincluding the orders Chytridiales, Ancylistales, Blastocladiales,Monoblepharidales, Saprolegniales, Peronosporales, Mucorales, andEntomophthorales.

In the Protozoa: Protozoa strains with life stages capable ofbacterivory (including by phageocytosis) can be selected from the typesclassified as ciliates, flagellates or amoebae. Protozoan ciliatesinclude the groups: Chonotrichs, Colpodids, Cyrtophores, Haptorids,Karyorelicts, Oligohymenophora, Polyhymenophora (spirotrichs), Prostomesand Suctoria. Protozoan flagellates include the Biosoecids, Bodonids,Cercomonads, Chrysophytes (for example the genera Anthophysa,Chrysamoemba, Chrysosphaerella, Dendromonas, Dinobryon, Mallomonas,Ochromonas, Paraphysomonas, Poterioochromonas, Spumella, Syncrypta,Synura, and Uroglena), Collar flagellates, Cryptophytes (for example thegenera Chilomonas, Cryptomonas, Cyanomonas, and Goniomonas),Dinoflagellates, Diplomonads, Euglenoids, Heterolobosea, Pedinellids,Pelobionts, Phalansteriids, Pseudodendromonads, Spongomonads andVolvocales (and other flagellates including the unassigned flagellategenera of Artodiscus, Clautriavia, Helkesimastix, Kathablepharis andMulticilia). Amoeboid protozoans include the groups: Actinophryids,Centrohelids, Desmothoricids, Diplophryids, Eumamoebae, Heterolobosea,Leptomyxids, Nucleariid filose amoebae, Pelebionts, Testate amoebae andVampyrellids (and including the unassigned amoebid genera Gymnophrys,Biomyxa, Microcometes, Reticulomyxa, Belonocystis, Elaeorhanis,Allelogromia, Gromia or Lieberkuhnia). The protozoan orders include thefollowing: Percolomonadeae, Heterolobosea, Lyromonadea, Pseudociliata,Trichomonadea, Hypermastigea, Heteromiteae, Telonemea, Cyathobodonea,Ebridea, Pyytomyxea, Opalinea, Kinetomonadea, Hemimastigea, Protostelea,Myxagastrea, Dictyostelea, Choanomonadea, Apicomonadea, Eogregarinea,Neogregarinea, Coelotrolphea, Eucoccidea, Haemosporea, Piroplasmea,Spirotrichea, Prostomatea, Litostomatea, Phyllopharyngea, Nassophorea,Oligohymenophorea, Colpodea, Karyorelicta, Nucleohelea, Centrohelea,Acantharea, Sticholonchea, Polycystinea, Phaeodarea, Lobosea, Filosea,Athalamea, Monothalamea, Polythalamea, Xenophyophorea, Schizocladea,Holosea, Entamoebea, Myxosporea, Actinomyxea, Halosporea, Paramyxea,Rhombozoa and Orthonectea.

A preferred embodiment of the present invention includes strains of themicroorganisms listed above that have been collected from one of thepreferred habitats listed above.

One embodiment of the present invention relates to any microorganismsidentified using the novel PUFA PKS screening method described above, tothe PUFA PKS genes and proteins encoded thereby, and to the use of suchmicroorganisms and/or PUFA PKS genes and proteins (including homologuesand fragments thereof) in any of the methods described herein. Inparticular, the present invention encompasses organisms identified bythe screening method of the present invention which are then geneticallymodified to regulate the production of bioactive molecules by said PUFAPKS system.

Yet another embodiment of the present invention relates to an isolatednucleic acid molecule comprising a nucleic acid sequence encoding atleast one biologically active domain or biologically active fragmentthereof of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS)system from a Thraustochytrid microorganism. As discussed above, thepresent inventors have successfully used the method to identify anon-bacterial microorganism that has a PUFA PKS system to identifyadditional members of the order Thraustochytriales which contain a PUFAPKS system. The identification of three such microorganisms is describedin Example 2. Specifically, the present inventors have used thescreening method of the present invention to identify Thraustochytriumsp. 23B (ATCC 20892) as being highly predicted to contain a PUFA PKSsystem, followed by detection of sequences in the Thraustochytrium sp.23B genome that hybridize to the Schizochytrium PUFA PKS genes disclosedherein. Schizochytrium limacium (IFO 32693) and Ulkenia (BP-5601) havealso been identified as good candidates for containing PUFA PKS systems.Based on these data and on the similarities among members of the orderThraustochytriales, it is believed that many other ThraustochytrialesPUFA PKS systems can now be readily identified using the methods andtools provided by the present invention. Therefore, ThraustochytrialesPUFA PKS systems and portions and/or homologues thereof (e.g., proteins,domains and fragments thereof), genetically modified organismscomprising such systems and portions and/or homologues thereof, andmethods of using such microorganisms and PUFA PKS systems, areencompassed by the present invention.

Developments have resulted in revision of the taxonomy of theThraustochytrids. Taxonomic theorists place Thraustochytrids with thealgae or algae-like protists. However, because of taxonomic uncertainty,it would be best for the purposes of the present invention to considerthe strains described in the present invention as Thraustochytrids(Order: Thraustochytriales; Family: Thraustochytriaceae; Genus:Thraustochytrium, Schizochytrium, Labyrinthuloides, or Japonochytrium).For the present invention, members of the labrinthulids are consideredto be included in the Thraustochytrids. Taxonomic changes are summarizedbelow. Strains of certain unicellular microorganisms disclosed hereinare members of the order Thraustochytriales. Thraustochytrids are marineeukaryotes with a evolving taxonomic history. Problems with thetaxonomic placement of the Thraustochytrids have been reviewed by Moss(1986), Bahnweb and Jackle (1986) and Chamberlain and Moss (1988).According to the present invention, the phrases “Thraustochytrid”,“Thraustochytriales microorganism” and “microorganism of the orderThraustochytriales” can be used interchangeably.

For convenience purposes, the Thraustochytrids were first placed bytaxonomists with other colorless zoosporic eukaryotes in thePhycomycetes (algae-like fungi). The name Phycomycetes, however, waseventually dropped from taxonomic status, and the Thraustochytrids wereretained in the Oomycetes (the biflagellate zoosporic fungi). It wasinitially assumed that the Oomycetes were related to the heterokontalgae, and eventually a wide range of ultrastructural and biochemicalstudies, summarized by Barr (Barr, 1981, Biosystems 14:359-370)supported this assumption. The Oomycetes were in fact accepted byLeedale (Leedale, 1974, Taxon 23:261-270) and other phycologists as partof the heterokont algae. However, as a matter of convenience resultingfrom their heterotrophic nature, the Oomycetes and Thraustochytrids havebeen largely studied by mycologists (scientists who study fungi) ratherthan phycologists (scientists who study algae).

From another taxonomic perspective, evolutionary biologists havedeveloped two general schools of thought as to how eukaryotes evolved.One theory proposes an exogenous origin of membrane-bound organellesthrough a series of endosymbioses (Margulis, 1970, Origin of EukarvoticCells. Yale University Press, New Haven); e.g., mitochondria werederived from bacterial endosymbionts, chloroplasts from cyanophytes, andflagella from spirochaetes. The other theory suggests a gradualevolution of the membrane-bound organelles from the non-membrane-boundedsystems of the prokaryote ancestor via an autogenous process(Cavalier-Smith, 1975, Nature (Lond.) 256:462-468). Both groups ofevolutionary biologists however, have removed the Oomycetes andThraustochytrids from the fungi and place them either with thechromophyte algae in the kingdom Chromophyta (Cavalier-Smith, 1981,BioSystems 14:461-481) (this kingdom has been more recently expanded toinclude other protists and members of this kingdom are now calledStramenopiles) or with all algae in the kingdom Protoctista (Margulisand Sagen, 1985, Biosystems 18:141-147).

With the development of electron microscopy, studies on theultrastructure of the zoospores of two genera of Thraustochytrids,Thraustochytrium and Schizochytrium, (Perkins, 1976, pp. 279-312 in“Recent Advances in Aquatic Mycology” (ed. E. B. G. Jones), John Wiley &Sons, New York; Kazama, 1980, Can. J. Bot. 58:2434-2446; Barr, 1981,Biosystems 14:359-370) have provided good evidence that theThraustochytriaceae are only distantly related to the Oomycetes.Additionally, genetic data representing a correspondence analysis (aform of multivariate statistics) of 5 S ribosomal RNA sequences indicatethat Thraustochytriales are clearly a unique group of eukaryotes,completely separate from the fungi, and most closely related to the redand brown algae, and to members of the Oomycetes (Mannella, et al.,1987, Mol. Evol. 24:228-235). Most taxonomists have agreed to remove theThraustochytrids from the Oomycetes (Bartnicki-Garcia, 1987, pp. 389-403in “Evolutionary Biology of the Fungi” (eds. Rayner, A. D. M., Brasier,C. M. & Moore, D.), Cambridge University Press, Cambridge).

In summary, employing the taxonomic system of Cavalier-Smith(Cavalier-Smith, 1981, BioSystems 14:461-481, 1983; Cavalier-Smith,1993, Microbiol Rev. 57:953-994), the Thraustochytrids are classifiedwith the chromophyte algae in the kingdom Chromophyta (Stramenopiles).This taxonomic placement has been more recently reaffirmed byCavalier-Smith et al. using the 18s rRNA signatures of the Heterokontato demonstrate that Thraustochytrids are chromists not Fungi(Cavalier-Smith et al., 1994, Phil. Tran. Roy. Soc. London SeriesBioSciences 346:387-397). This places them in a completely differentkingdom from the fungi, which are all placed in the kingdom Eufungi. Thetaxonomic placement of the Thraustochytrids is therefore summarizedbelow:

Kingdom: Chromophyta (Stramenopiles)

Phylum: Heterokonta

Order: Thraustochytriales

Family: Thraustochytriaceae

Genus: Thraustochytrium, Schizochytrium, Labyrinthuloides, orJaponochytrium

Some early taxonomists separated a few original members of the genusThraustochytrium (those with an amoeboid life stage) into a separategenus called Ulkenia. However it is now known that most, if not all,Thraustochytrids (including Thraustochytrium and Schizochytrium),exhibit amoeboid stages and as such, Ulkenia is not considered by someto be a valid genus. As used herein, the genus Thraustochytrium willinclude Ulkenia.

Despite the uncertainty of taxonomic placement within higherclassifications of Phylum and Kingdom, the Thraustochytrids remain adistinctive and characteristic grouping whose members remainclassifiable within the order Thraustochytriales.

Polyunsaturated fatty acids (PUFAs) are essential membrane components inhigher eukaryotes and the precursors of many lipid-derived signalingmolecules. The PUFA PKS system of the present invention uses pathwaysfor PUFA synthesis that do not require desaturation and elongation ofsaturated fatty acids. The pathways catalyzed by PUFA PKSs that aredistinct from previously recognized PKSs in both structure andmechanism. Generation of cis double bonds is suggested to involveposition-specific isomerases; these enzymes are believed to be useful inthe production of new families of antibiotics.

To produce significantly high yields of various bioactive moleculesusing the PUFA PKS system of the present invention, an organism,preferably a microorganism or a plant, can be genetically modified toaffect the activity of a PUFA PKS system. In one aspect, such anorganism can endogenously contain and express a PUFA PKS system, and thegenetic modification can be a genetic modification of one or more of thefunctional domains of the endogenous PUFA PKS system, whereby themodification has some effect on the activity of the PUFA PKS system. Inanother aspect, such an organism can endogenously contain and express aPUFA PKS system, and the genetic modification can be an introduction ofat least one exogenous nucleic acid sequence (e.g., a recombinantnucleic acid molecule), wherein the exogenous nucleic acid sequenceencodes at least one biologically active domain or protein from a secondPKS system and/or a protein that affects the activity of said PUFA PKSsystem (e.g., a phosphopantetheinyl transferases (PPTase), discussedbelow). In yet another aspect, the organism does not necessarilyendogenously (naturally) contain a PUFA PKS system, but is geneticallymodified to introduce at least one recombinant nucleic acid moleculeencoding an amino acid sequence having the biological activity of atleast one domain of a PUFA PKS system. In this aspect, PUFA PKS activityis affected by introducing or increasing PUFA PKS activity in theorganism. Various embodiments associated with each of these aspects willbe discussed in greater detail below.

Therefore, according to the present invention, one embodiment relates toa genetically modified microorganism, wherein the microorganismexpresses a PKS system comprising at least one biologically activedomain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS)system. The at least one domain of the PUFA PKS system is encoded by anucleic acid sequence chosen from: (a) a nucleic acid sequence encodingat least one domain of a polyunsaturated fatty acid (PUFA) polyketidesynthase (PKS) system from a Thraustochytrid microorganism; (b) anucleic acid sequence encoding at least one domain of a PUFA PKS systemfrom a microorganism identified by a screening method of the presentinvention; (c) a nucleic acid sequence encoding an amino acid sequencethat is at least about 60% identical to at least 500 consecutive aminoacids of an amino acid sequence selected from the group consisting of:SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6; wherein the amino acidsequence has a biological activity of at least one domain of a PUFA PKSsystem; and, (d) a nucleic acid sequence encoding an amino acid sequencethat is at least about 60% identical to an amino acid sequence selectedfrom the group consisting of: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13,SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26,SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:32; wherein the amino acidsequence has a biological activity of at least one domain of a PUFA PKSsystem. The genetic modification affects the activity of the PKS systemin the organism. The screening process referenced in part (b) has beendescribed in detail above and includes the steps of: (a) selecting amicroorganism that produces at least one PUFA; and, (b) identifying amicroorganism from (a) that has an ability to produce increased PUFAsunder dissolved oxygen conditions of less than about 5% of saturation inthe fermentation medium, as compared to production of PUFAs by themicroorganism under dissolved oxygen conditions of greater than about 5%of saturation, and preferably about 10%, and more preferably about 15%,and more preferably about 20% of saturation in the fermentation medium.The genetically modified microorganism can include anyone or more of theabove-identified nucleic acid sequences, and/or any of the otherhomologues of any of the Schizochytrium PUFA PKS ORFs or domains asdescribed in detail above.

As used herein, a genetically modified microorganism can include agenetically modified bacterium, protist, microalgae, fungus, or othermicrobe, and particularly, any of the genera of the orderThraustochytriales (e.g., a Thraustochytrid) described herein (e.g.,Schizochytrium, Thraustochytrium, Japonochytrium, Labyrinthuloides).Such a genetically modified microorganism has a genome which is modified(i.e., mutated or changed) from its normal (i.e., wild-type or naturallyoccurring) form such that the desired result is achieved (i.e.,increased or modified PUFA PKS activity and/or production of a desiredproduct using the PKS system). Genetic modification of a microorganismcan be accomplished using classical strain development and/or moleculargenetic techniques. Such techniques known in the art and are generallydisclosed for microorganisms, for example, in Sambrook et al., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press.The reference Sambrook et al., ibid., is incorporated by referenceherein in its entirety. A genetically modified microorganism can includea microorganism in which nucleic acid molecules have been inserted,deleted or modified (i.e., mutated; e.g., by insertion, deletion,substitution, and/or inversion of nucleotides), in such a manner thatsuch modifications provide the desired effect within the microorganism.

Preferred microorganism host cells to modify according to the presentinvention include, but are not limited to, any bacteria, protist,microalga, fungus, or protozoa. In one aspect, preferred microorganismsto genetically modify include, but are not limited to, any microorganismof the order Thraustochytriales. Particularly preferred host cells foruse in the present invention could include microorganisms from a genusincluding, but not limited to: Thraustochytrium, Labyrinthuloides,Japonochytrium, and Schizochytrium. Preferred species within thesegenera include, but are not limited to: any Schizochytrium species,including Schizochytrium aggregatum, Schizochytrium limacinum,Schizochytrium minutum; any Thraustochytrium species (including formerUlkenia species such as U. visurgensis, U. amoeboida, U. sarkariana, U.profunda, U. radiata, U. minuta and Ulkenia sp. BP-5601), and includingThraustochytrium striatum, Thraustochytrium aureum, Thraustochytriumroseum; and any Japonochytrium species. Particularly preferred strainsof Thraustochytriales include, but are not limited to: Schizochytriumsp. (S31) (ATCC 20888); Schizochytrium sp. (S8) (ATCC 20889);Schizochytrium sp. (LC-RM) (ATCC 18915); Schizochytrium sp. (SR21);Schizochytrium aggregatum (Goldstein et Belsky) (ATCC 28209);Schizochytrium limacinum (Honda et Yokochi) (IFO 32693);Thraustochytrium sp. (23B) (ATCC 20891); Thraustochytrium striatum(Schneider) (ATCC 24473); Thraustochytrium aureum (Goldstein) (ATCC34304); Thraustochytrium roseum (Goldstein) (ATCC 28210); andJaponochytrium sp. (L1) (ATCC 28207). Other examples of suitable hostmicroorganisms for genetic modification include, but are not limited to,yeast including Saccharomyces cerevisiae, Saccharomyces carlsbergensis,or other yeast such as Candida, Kluyveromyces, or other fungi, forexample, filamentous fungi such as Aspergillus, Neurospora, Penicillium,etc. Bacterial cells also may be used as hosts. This includesEscherichia coli, which can be useful in fermentation processes.Alternatively, a host such as a Lactobacillus species or Bacillusspecies can be used as a host.

Another embodiment of the present invention relates to a geneticallymodified plant, wherein the plant has been genetically modified torecombinantly express a PKS system comprising at least one biologicallyactive domain of a polyunsaturated fatty acid (PUFA) polyketide synthase(PKS) system. The domain is encoded by a nucleic acid sequence chosenfrom: (a) a nucleic acid sequence encoding at least one domain of apolyunsaturated fatty acid (PUFA) polyketide synthase (PKS) system froma Thraustochytrid microorganism; (b) a nucleic acid sequence encoding atleast one domain of a PUFA PKS system from a microorganism identified bythe screening and selection method described herein (see brief summaryof method in discussion of genetically modified microorganism above);(c) a nucleic acid sequence encoding an amino acid sequence selectedfrom the group consisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, andbiologically active fragments thereof; (d) a nucleic acid sequenceencoding an amino acid sequence selected from the group consisting of:SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:20, SEQID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, and biologically active fragments thereof, (e) a nucleic acidsequence encoding an amino acid sequence that is at least about 60%identical to at least 500 consecutive amino acids of an amino acidsequence selected from the group consisting of: SEQ ID NO:2, SEQ IDNO:4, and SEQ ID NO:6; wherein the amino acid sequence has a biologicalactivity of at least one domain of a PUFA PKS system; and/or (f) anucleic acid sequence encoding an amino acid sequence that is at leastabout 60% identical to an amino acid sequence selected from the groupconsisting of: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28,SEQ ID NO:30, and SEQ ID NO:32; wherein the amino acid sequence has abiological activity of at least one domain of a PUFA PKS system. Thegenetically modified plant can include any one or more of theabove-identified nucleic acid sequences, and/or any of the otherhomologues of any of the Schizochytrium PUFA PKS ORFs or domains asdescribed in detail above.

As used herein, a genetically modified plant can include any geneticallymodified plant including higher plants and particularly, any consumableplants or plants useful for producing a desired bioactive molecule ofthe present invention. Such a genetically modified plant has a genomewhich is modified (i.e., mutated or changed) from its normal (i.e.,wild-type or naturally occurring) form such that the desired result isachieved (i.e., increased or modified PUFA PKS activity and/orproduction of a desired product using the PKS system). Geneticmodification of a plant can be accomplished using classical straindevelopment and/or molecular genetic techniques. Methods for producing atransgenic plant, wherein a recombinant nucleic acid molecule encoding adesired amino acid sequence is incorporated into the genome of theplant, are known in the art. A preferred plant to genetically modifyaccording to the present invention is preferably a plant suitable forconsumption by animals, including humans.

Preferred plants to genetically modify according to the presentinvention (i.e., plant host cells) include, but are not limited to anyhigher plants, and particularly consumable plants, including crop plantsand especially plants used for their oils. Such plants can include, forexample: canola, soybeans, rapeseed, linseed, corn, safflowers,sunflowers and tobacco. Other preferred plants include those plants thatare known to produce compounds used as pharmaceutical agents, flavoringagents, neutraceutical agents, functional food ingredients orcosmetically active agents or plants that are genetically engineered toproduce these compounds/agents.

According to the present invention, a genetically modified microorganismor plant includes a microorganism or plant that has been modified usingrecombinant technology. As used herein, genetic modifications whichresult in a decrease in gene expression, in the function of the gene, orin the function of the gene product (i.e., the protein encoded by thegene) can be referred to as inactivation (complete or partial),deletion, interruption, blockage or down-regulation of a gene. Forexample, a genetic modification in a gene which results in a decrease inthe function of the protein encoded by such gene, can be the result of acomplete deletion of the gene (i.e., the gene does not exist, andtherefore the protein does not exist), a mutation in the gene whichresults in incomplete or no translation of the protein (e.g., theprotein is not expressed), or a mutation in the gene which decreases orabolishes the natural function of the protein (e.g., a protein isexpressed which has decreased or no enzymatic activity or action).Genetic modifications that result in an increase in gene expression orfunction can be referred to as amplification, overproduction,overexpression, activation, enhancement, addition, or up-regulation of agene.

The genetic modification of a microorganism or plant according to thepresent invention preferably affects the activity of the PKS systemexpressed by the plant, whether the PKS system is endogenous andgenetically modified, endogenous with the introduction of recombinantnucleic acid molecules into the organism, or provided completely byrecombinant technology. According to the present invention, to “affectthe activity of a PKS system” includes any genetic modification thatcauses any detectable or measurable change or modification in the PKSsystem expressed by the organism as compared to in the absence of thegenetic modification. A detectable change or modification in the PKSsystem can include, but is not limited to: the introduction of PKSsystem activity into an organism such that the organism now hasmeasurable/detectable PKS system activity (i.e., the organism did notcontain a PKS system prior to the genetic modification), theintroduction into the organism of a functional domain from a differentPKS system than a PKS system endogenously expressed by the organism suchthat the PKS system activity is modified (e.g., a bacterial PUFA PKSdomain or a type I PKS domain is introduced into an organism thatendogenously expresses a non-bacterial PUFA PKS system), a change in theamount of a bioactive molecule produced by the PKS system (e.g., thesystem produces more (increased amount) or less (decreased amount) of agiven product as compared to in the absence of the geneticmodification), a change in the type of a bioactive molecule produced bythe PKS system (e.g., the system produces a new or different product, ora variant of a product that is naturally produced by the system), and/ora change in the ratio of multiple bioactive molecules produced by thePKS system (e.g., the system produces a different ratio of one PUFA toanother PUFA, produces a completely different lipid profile as comparedto in the absence of the genetic modification, or places various PUFAsin different positions in a triacylglycerol as compared to the naturalconfiguration). Such a genetic modification includes any type of geneticmodification and specifically includes modifications made by recombinanttechnology and by classical mutagenesis.

It should be noted that reference to increasing the activity of afunctional domain or protein in a PUFA PKS system refers to any geneticmodification in the organism containing the domain or protein (or intowhich the domain or protein is to be introduced) which results inincreased functionality of the domain or protein system and can includehigher activity of the domain or protein (e.g., specific activity or invivo enzymatic activity), reduced inhibition or degradation of thedomain or protein system, and overexpression of the domain or protein.For example, gene copy number can be increased, expression levels can beincreased by use of a promoter that gives higher levels of expressionthan that of the native promoter, or a gene can be altered by geneticengineering or classical mutagenesis to increase the activity of thedomain or protein encoded by the gene.

Similarly, reference to decreasing the activity of a functional domainor protein in a PUFA PKS system refers to any genetic modification inthe organism containing such domain or protein (or into which the domainor protein is to be introduced) which results in decreased functionalityof the domain or protein and includes decreased activity of the domainor protein, increased inhibition or degradation of the domain or proteinand a reduction or elimination of expression of the domain or protein.For example, the action of domain or protein of the present inventioncan be decreased by blocking or reducing the production of the domain orprotein, “knocking out” the gene or portion thereof encoding the domainor protein, reducing domain or protein activity, or inhibiting theactivity of the domain or protein. Blocking or reducing the productionof an domain or protein can include placing the gene encoding the domainor protein under the control of a promoter that requires the presence ofan inducing compound in the growth medium. By establishing conditionssuch that the inducer becomes depleted from the medium, the expressionof the gene encoding the domain or protein (and therefore, of proteinsynthesis) could be turned off. Blocking or reducing the activity ofdomain or protein could also include using an excision technologyapproach similar to that described in U.S. Pat. No. 4,743,546,incorporated herein by reference. To use this approach, the geneencoding the protein of interest is cloned between specific geneticsequences that allow specific, controlled excision of the gene from thegenome. Excision could be prompted by, for example, a shift in thecultivation temperature of the culture, as in U.S. Pat. No. 4,743,546,or by some other physical or nutritional signal.

In one embodiment of the present invention, a genetic modificationincludes a modification of a nucleic acid sequence encoding an aminoacid sequence that has a biological activity of at least one domain of anon-bacterial PUFA PKS system as described herein. Such a modificationcan be to an amino acid sequence within an endogenously (naturally)expressed non-bacterial PUFA PKS system, whereby a microorganism thatnaturally contains such a system is genetically modified by, forexample, classical mutagenesis and selection techniques and/or moleculargenetic techniques, include genetic engineering techniques. Geneticengineering techniques can include, for example, using a targetingrecombinant vector to delete a portion of an endogenous gene, or toreplace a portion of an endogenous gene with a heterologous sequence.Examples of heterologous sequences that could be introduced into a hostgenome include sequences encoding at least one functional domain fromanother PKS system, such as a different non-bacterial PUFA PKS system, abacterial PUFA PKS system, a type I PKS system, a type II PKS system, ora modular PKS system. Other heterologous sequences to introduce into thegenome of a host includes a sequence encoding a protein or functionaldomain that is not a domain of a PKS system, but which will affect theactivity of the endogenous PKS system. For example, one could introduceinto the host genome a nucleic acid molecule encoding aphosphopantetheinyl transferase (discussed below). Specificmodifications that could be made to an endogenous PUFA PKS system arediscussed in detail below.

In another aspect of this embodiment of the invention, the geneticmodification can include: (1) the introduction of a recombinant nucleicacid molecule encoding an amino acid sequence having a biologicalactivity of at least one domain of a non-bacterial PUFA PKS system;and/or (2) the introduction of a recombinant nucleic acid moleculeencoding a protein or functional domain that affects the activity of aPUFA PKS system, into a host. The host can include: (1) a host cell thatdoes not express any PKS system, wherein all functional domains of a PKSsystem are introduced into the host cell, and wherein at least onefunctional domain is from a non-bacterial PUFA PKS system; (2) a hostcell that expresses a PKS system (endogenous or recombinant) having atleast one functional domain of a non-bacterial PUFA PKS system, whereinthe introduced recombinant nucleic acid molecule can encode at least oneadditional non-bacterial PUFA PKS domain function or another protein ordomain that affects the activity of the host PKS system; and (3) a hostcell that expresses a PKS system (endogenous or recombinant) which doesnot necessarily include a domain function from a non-bacterial PUFA PKS,and wherein the introduced recombinant nucleic acid molecule includes anucleic acid sequence encoding at least one functional domain of anon-bacterial PUFA PKS system. In other words, the present inventionintends to encompass any genetically modified organism (e.g.,microorganism or plant), wherein the organism comprises at least onenon-bacterial PUFA PKS domain function (either endogenously or byrecombinant modification), and wherein the genetic modification has ameasurable effect on the non-bacterial PUFA PKS domain function or onthe PKS system when the organism comprises a functional PKS system.

Therefore, using the non-bacterial PUFA PKS systems of the presentinvention, which, for example, makes use of genes from ThraustochytridPUFA PKS systems, gene mixing can be used to extend the range of PUFAproducts to include EPA, DHA, ARA, GLA, SDA and others, as well as toproduce a wide variety of bioactive molecules, including antibiotics,other pharmaceutical compounds, and other desirable products. The methodto obtain these bioactive molecules includes not only the mixing ofgenes from various organisms but also various methods of geneticallymodifying the non-bacterial PUFA PKS genes disclosed herein. Knowledgeof the genetic basis and domain structure of the non-bacterial PUFA PKSsystem of the present invention provides a basis for designing novelgenetically modified organisms which produce a variety of bioactivemolecules. Although mixing and modification of any PKS domains andrelated genes are contemplated by the present inventors, by way ofexample, various possible manipulations of the PUFA-PKS system arediscussed below with regard to genetic modification and bioactivemolecule production.

For example, in one embodiment, non-bacterial PUFA-PKS system products,such as those produced by Thraustochytrids, are altered by modifying theCLF (chain length factor) domain. This domain is characteristic of TypeII (dissociated enzymes) PKS systems. Its amino acid sequence showshomology to KS (keto synthase pairs) domains, but it lacks the activesite cysteine. CLF may function to determine the number of elongationcycles, and hence the chain length, of the end product. In thisembodiment of the invention, using the current state of knowledge of FASand PKS synthesis, a rational strategy for production of ARA by directedmodification of the non-bacterial PUFA-PKS system is provided. There iscontroversy in the literature concerning the function of the CLF in PKSsystems (C. Bisang et al., Nature 401, 502 (1999)) and it is realizedthat other domains may be involved in determination of the chain lengthof the end product. However, it is significant that Schizochytriumproduces both DHA (C22:6, ω-3) and DPA (C22:5, ω-6). In the PUFA-PKSsystem the cis double bonds are introduced during synthesis of thegrowing carbon chain. Since placement of the ω-3 and ω-6 double bondsoccurs early in the synthesis of the molecules, one would not expectthat they would affect subsequent end-product chain lengthdetermination. Thus, without being bound by theory, the presentinventors believe that introduction of a factor (e.g. CLF) that directssynthesis of C20 units (instead of C22 units) into the SchizochytriumPUFA-PKS system will result in the production of EPA (C20:5, ω-3) andARA (C20:4, ω-6). For example, in heterologous systems, one couldexploit the CLF by directly substituting a CLF from an EPA producingsystem (such as one from Photobacterium) into the Schizochytrium geneset. The fatty acids of the resulting transformants can then be analyzedfor alterations in profiles to identify the transformants producing EPAand/or ARA.

In addition to dependence on development of a heterologous system(recombinant system, such as could be introduced into plants), the CLFconcept can be exploited in Schizochytrium (i.e., by modification of aSchizochytrium genome). Transformation and homologous recombination hasbeen demonstrated in Schizochytrium. One can exploit this byconstructing a clone with the CLF of OrfB replaced with a CLF from a C20PUFA-PKS system. A marker gene will be inserted downstream of the codingregion. One can then transform the wild type cells, select for themarker phenotype and then screen for those that had incorporated the newCLF. Again, one would analyze these for any effects on fatty acidprofiles to identify transformants producing EPA and/or ARA. If somefactor other than those associated with the CLF are found to influencethe chain length of the end product, a similar strategy could beemployed to alter those factors.

Another preferred embodiment involving alteration of the PUFA-PKSproducts involves modification or substitution of the β-hydroxy acyl-ACPdehydrase/keto synthase pairs. During cis-vaccenic acid (C18:1, Δ11)synthesis in E. coli, creation of the cis double bond is believed todepend on a specific DH enzyme, β-hydroxy acyl-ACP dehydrase, theproduct of the FabA gene. This enzyme removes HOH from a β-keto acyl-ACPand leaves a trans double bond in the carbon chain. A subset of DH's,FabA-like, possess cis-trans isomerase activity (Heath et al., 1996,supra). A novel aspect of bacterial and non-bacterial PUFA-PKS systemsis the presence of two FabA-like DH domains. Without being bound bytheory, the present inventors believe that one or both of these DHdomains will possess cis-trans isomerase activity (manipulation of theDH domains is discussed in greater detail below).

Another aspect of the unsaturated fatty acid synthesis in E. coli is therequirement for a particular KS enzyme, β-ketoacyl-ACP synthase, theproduct of the FabB gene. This is the enzyme that carries outcondensation of a fatty acid, linked to a cysteine residue at the activesite (by a thio-ester bond), with a malonyl-ACP. In the multi-stepreaction, CO₂ is released and the linear chain is extended by twocarbons. It is believed that only this KS can extend a carbon chain thatcontains a double bond. This extension occurs only when the double bondis in the cis configuration; if it is in the trans configuration, thedouble bond is reduced by enoyl-ACP reductase (ER) prior to elongation(Heath et al., 1996, supra). All of the PUFA-PKS systems characterizedso far have two KS domains, one of which shows greater homology to theFabB-like KS of E. coli than the other. Again, without being bound bytheory, the present inventors believe that in PUFA-PKS systems, thespecificities and interactions of the DH (FabA-like) and KS (FabB-like)enzymatic domains determine the number and placement of cis double bondsin the end products. Because the number of 2-carbon elongation reactionsis greater than the number of double bonds present in the PUFA-PKS endproducts, it can be determined that in some extension cycles completereduction occurs. Thus the DH and KS domains can be used as targets foralteration of the DHA/DPA ratio or ratios of other long chain fattyacids. These can be modified and/or evaluated by introduction ofhomologous domains from other systems or by mutagenesis of these genefragments.

In another embodiment, the ER (enoyl-ACP reductase—an enzyme whichreduces the trans-double bond in the fatty acyl-ACP resulting in fullysaturated carbons) domains can be modified or substituted to change thetype of product made by the PKS system. For example, the presentinventors know that Schizochytrium PUFA-PKS system differs from thepreviously described bacterial systems in that it has two (rather thanone) ER domains. Without being bound by theory, the present inventorsbelieve these ER domains can strongly influence the resulting PKSproduction product. The resulting PKS product could be changed byseparately knocking out the individual domains or by modifying theirnucleotide sequence or by substitution of ER domains from otherorganisms.

In another embodiment, nucleic acid molecules encoding proteins ordomains that are not part of a PKS system, but which affect a PKSsystem, can be introduced into an organism. For example, all of the PUFAPKS systems described above contain multiple, tandem, ACP domains. ACP(as a separate protein or as a domain of a larger protein) requiresattachment of a phosphopantetheine cofactor to produce the active,holo-ACP. Attachment of phosphopantetheine to the apo-ACP is carried outby members of the superfamily of enzymes—the phosphopantetheinyltransferases (PPTase) (Lambalot R. H., et al., Chemistry and Biology, 3,923 (1996)).

By analogy to other PKS and FAS systems, the present inventors presumethat activation of the multiple ACP domains present in theSchizochytrium ORFA protein is carried out by a specific, endogenous,PPTase. The gene encoding this presumed PPTase has not yet beenidentified in Schizochytrium. If such a gene is present inSchizochytrium, one can envision several approaches that could be usedin an attempt to identify and clone it. These could include (but wouldnot be limited to): generation and partial sequencing of a cDNA libraryprepared from actively growing Schizochytrium cells (note, one sequencewas identified in the currently available Schizochytrium cDNA libraryset which showed homology to PPTase's; however, it appears to be part ofa multidomain FAS protein, and as such may not encode the desired OrfAspecific PPTase); use of degenerate oligonucleotide primers designedusing amino acid motifs present in many PPTase's in PCR reactions (toobtain a nucleic acid probe molecule to screen genomic or cDNAlibraries); genetic approaches based on protein-protein interactions(e.g. a yeast two-hybrid system) in which the ORFA-ACP domains would beused as a “bait” to find a “target” (i.e. the PPTase); and purificationand partial sequencing of the enzyme itself as a means to generate anucleic acid probe for screening of genomic or cDNA libraries.

It is also conceivable that a heterologous PPTase may be capable ofactivating the Schizochytrium ORFA ACP domains. It has been shown thatsome PPTases, for example the sfp enzyme of Bacillus subtilis (Lambalotet al., supra) and the svp enzyme of Streptomyces verticillus (Sanchezet al., 2001, Chemistry & Biology 8:725-738), have a broad substratetolerance. These enzymes can be tested to see if they will activate theSchizochytrium ACP domains. Also, a recent publication described theexpression of a fungal PKS protein in tobacco (Yalpani et al., 2001, ThePlant Cell 13:1401-1409). Products of the introduced PKS system (encodedby the 6-methylsalicyclic acid synthase gene of Penicillium patulum)were detected in the transgenic plant, even though the correspondingfungal PPTase was not present in those plants. This suggested that anendogenous plant PPTase(s) recognized and activated the fungal PKS ACPdomain. Of relevance to this observation, the present inventors haveidentified two sequences (genes) in the Arabidopsis whole genomedatabase that are likely to encode PPTases. These sequences (GenBankAccession numbers; AAG51443 and AAC05345) are currently listed asencoding “Unknown Proteins”. They can be identified as putative PPTasesbased on the presence in the translated protein sequences of severalsignature motifs including; G(I/V)D and WxxKE(A/S)xxK (SEQ ID NO:33),(listed in Lambalot et al., 1996 as characteristic of all PPTases). Inaddition, these two putative proteins contain two additional motifstypically found in PPTases typically associated with PKS andnon-ribosomal peptide synthesis systems; i.e., FN(I/L/V)SHS (SEQ IDNO:34) and (I/V/L)G(I/L/V)D(I/L/V) (SEQ ID NO:35). Furthermore, thesemotifs occur in the expected relative positions in the proteinsequences. It is likely that homologues of the Arabidopsis genes arepresent in other plants, such as tobacco. Again, these genes can becloned and expressed to see if the enzymes they encode can activate theSchizochytrium ORFA ACP domains, or alternatively, OrfA could beexpressed directly in the transgenic plant (either targeted to theplastid or the cytoplasm).

Another heterologous PPTase which may recognize the ORFA ACP domains assubstrates is the Het I protein of Nostoc sp. PCC 7120 (formerly calledAnabaena sp. PCC 7120). As noted in U.S. Pat. No. 6,140,486, several ofthe PUFA-PKS genes of Shewanella showed a high degree of homology toprotein domains present in a PKS cluster found in Nostoc (FIG. 2 of thatpatent). This Nostoc PKS system is associated with the synthesis of longchain (C26 or C28) hydroxy fatty acids that become esterified to sugarmoieties and form a part of the heterocyst cell wall. These Nostoc PKSdomains are also highly homologous to the domains found in Orfs B and Cof the Schizochytrium PKS proteins (i.e. the same ones that correspondto those found in the Shewanella PKS proteins). Until very recently,none of the Nostoc PKS domains present in the GenBank databases showedhigh homology to any of the domains of Schizochytrium OrfA (or thehomologous Shewanella Orf 5 protein). However, the complete genome ofNostoc has recently been sequenced and as a result, the sequence of theregion just upstream of the PKS gene cluster is now available. In thisregion are three Orfs that show homology to the domains (KS, MAT, ACPand KR) of OrfA (see FIG. 3). Included in this set are two ACP domains,both of which show high homology to the ORFA ACP domains. At the end ofthe Nostoc PKS cluster is the gene that encodes the Het I PPTase.Previously, it was not obvious what the substrate of the Het I enzymecould be, however the presence of tandem ACP domains in the newlyidentified Orf (Hgl E) of the cluster strongly suggests to the presentinventors that it is those ACPs. The homology of the ACP domains ofSchizochytrium and Nostoc, as well as the tandem arrangement of thedomains in both proteins, makes Het I a likely candidate forheterologous activation of the Schizochytrium ORFA ACPs. The presentinventors are believed to be the first to recognize and contemplate thisuse for Nostoc Het I PPTase.

As indicated in Metz et al., 2001, supra, one novel feature of the PUFAPKS systems is the presence of two dehydratase domains, both of whichshow homology to the FabA proteins of E. coli. With the availability ofthe new Nostoc PKS gene sequences mentioned above, one can now comparethe two systems and their products. The sequence of domains in theNostoc cluster (from HglE to Het I) as the present inventors havedefined them is (see FIG. 3):

KS-MAT-2×ACP, KR, KS, CLF-AT, ER (HetM, HetN) HetI

In the Schizochytrium PUFA-PKS Orfs A, B&C the sequence (OrfA-B-C) is:

KS-MAT-9×ACP-KR KS-CLF-AT-ER DH-DH-ER

One can see the correspondence of the domains sequence (there is also ahigh amino acid sequence homology). The product of the Nostoc PKS systemis a long chain hydroxy fatty acid (C26 or C28 with one or two hydroxygroups) that contains no double bonds (cis or trans). The product of theSchizochytrium PKS system is a long chain polyunsaturated fatty acid(C22, with 5 or 6 double bonds—all cis). An obvious difference betweenthe two domain sets is the presence of the two DH domains in theSchizochytrium proteins—just the domains implicated in the formation ofthe cis double bonds of DHA and DPA (presumably HetM and HetN in theNostoc system are involved in inclusion of the hydroxyl groups and alsocontain a DH domain whose origin differs from the those found in thePUFA). Also, the role of the duplicated ER domain in the SchizochytriumOrfs B and C is not known (the second ER domain in is not present othercharacterized PUFA PKS systems). The amino acid sequence homologybetween the two sets of domains implies an evolutionary relationship.One can conceive of the PUFA PKS gene set being derived from (in anevolutionary sense) an ancestral Nostoc-like PKS gene set byincorporation of the DH (FabA-like) domains. The addition of the DHdomains would result in the introduction of cis double bonds in the newPKS end product structure.

The comparisons of the Schizochytrium and Nostoc PKS domain structuresas well as the comparison of the domain organization between theSchizochytrium and Shewanella PUFA-PKS proteins demonstrate nature'sability to alter domain order as well as incorporate new domains tocreate novel end products. In addition, the genes can now be manipulatedin the laboratory to create new products. The implication from theseobservations is that it should be possible to continue to manipulate thesystems in either a directed or random way to influence the endproducts. For example, in a preferred embodiment, one could envisionsubstituting one of the DH (FabA-like) domains of the PUFA-PKS systemfor a DH domain that did not posses isomerization activity, potentiallycreating a molecule with a mix of cis- and trans-double bonds. Thecurrent products of the Schizochytrium PUFA PKS system are DHA and DPA(C22:5 ω6). If one manipulated the system to produce C20 fatty acids,one would expect the products to be EPA and ARA (C20:4 ω6). This couldprovide a new source for ARA. One could also substitute domains fromrelated PUFA-PKS systems that produced a different DHA to DPA ratio—forexample by using genes from Thraustochytrium 23B (the PUFA PKS system ofwhich is identified for the first time herein).

Additionally, one could envision specifically altering one of the ERdomains (e.g. removing, or inactivating) in the Schizochytrium PUFA PKSsystem (other PUFA PKS systems described so far do not have two ERdomains) to determine its effect on the end product profile. Similarstrategies could be attempted in a directed manner for each of thedistinct domains of the PUFA-PKS proteins using more or lesssophisticated approaches. Of course one would not be limited to themanipulation of single domains. Finally, one could extend the approachby mixing domains from the PUFA-PKS system and other PKS or FAS systems(e.g., type I, type II, modular) to create an entire range of new endproducts. For example, one could introduce the PUFA-PKS DH domains intosystems that do not normally incorporate cis double bonds into their endproducts.

Accordingly, encompassed by the present invention are methods togenetically modify microbial or plant cells by: genetically modifying atleast one nucleic acid sequence in the organism that encodes an aminoacid sequence having the biological activity of at least one functionaldomain of a non-bacterial PUFA PKS system according to the presentinvention, and/or expressing at least one recombinant nucleic acidmolecule comprising a nucleic acid sequence encoding such amino acidsequence. Various embodiments of such sequences, methods to geneticallymodify an organism, and specific modifications have been described indetail above. Typically, the method is used to produce a particulargenetically modified organism that produces a particular bioactivemolecule or molecules.

One embodiment of the present invention relates to a recombinant hostcell which has been modified to express a polyunsaturated fatty acid(PUFA) polyketide synthase (PKS) system, wherein the PKS catalyzes bothiterative and non-iterative enzymatic reactions, and wherein the PUFAPKS system comprises: (a) at least two enoyl ACP-reductase (ER) domains;(b) at least six acyl carrier protein (ACP) domains; (c) at least twoβ-keto acyl-ACP synthase (KS) domains; (d) at least one acyltransferase(AT) domain; (e) at least one ketoreductase (KR) domain; (f) at leasttwo FabA-like β-hydroxy acyl-ACP dehydrase (DH) domains; (g) at leastone chain length factor (CLF) domain; and (h) at least onemalonyl-CoA:ACP acyltransferase (MAT) domain. In one embodiment, thePUFA PKS system is a eukaryotic PUFA PKS system. In a preferredembodiment, the PUFA PKS system is an algal PUFA PKS system. In a morepreferred embodiment, the PUFA PKS system is a Thraustochytriales PUFAPKS system. Such PUFA PKS systems can include, but are not limited to, aSchizochytrium PUFA PKS system, and a Thraustochytrium PUFA PKS system.In one embodiment, the PUFA PKS system can be expressed in a prokaryotichost cell. In another embodiment, the PUFA PKS system can be expressedin a eukaryotic host cell.

Another embodiment of the present invention relates to a recombinanthost cell which has been modified to express a non-bacterial PUFA PKSsystem, wherein the PKS system catalyzes both iterative andnon-iterative enzymatic reactions, and wherein the non-bacterial PUFAPKS system comprises at least the following biologically active domains:(a) at least one enoyl ACP-reductase (ER) domain; (b) multiple acylcarrier protein (ACP) domains (at least four); (c) at least two β-ketoacyl-ACP synthase (KS) domains; (d) at least one acyltransferase (AT)domain; (e) at least one ketoreductase (KR) domain; (f) at least twoFabA-like β-hydroxy acyl-ACP dehydrase (DH) domains; (g) at least onechain length factor (CLF) domain; and (h) at least one malonyl-CoA:ACPacyltransferase (MAT) domain.

One aspect of this embodiment of the invention relates to a method toproduce a product containing at least one PUFA, comprising growing aplant comprising any of the recombinant host cells described above,wherein the recombinant host cell is a plant cell, under conditionseffective to produce the product. Another aspect of this embodiment ofthe invention relates to a method to produce a product containing atleast one PUFA, comprising culturing a culture containing any of therecombinant host cells described above, wherein the host cell is amicrobial cell, under conditions effective to produce the product. In apreferred embodiment, the PKS system in the host cell catalyzes thedirect production of triglycerides.

Another embodiment of the present invention relates to a microorganismcomprising a non-bacterial, polyunsaturated fatty acid (PUFA) polyketidesynthase (PKS) system, wherein the PKS catalyzes both iterative andnon-iterative enzymatic reactions, and wherein the PUFA PKS systemcomprises: (a) at least two enoyl ACP-reductase (ER) domains; (b) atleast six acyl carrier protein (ACP) domains; (c) at least two β-ketoacyl-ACP synthase (KS) domains; (d) at least one acyltransferase (AT)domain; (e) at least one ketoreductase (KR) domain; (f) at least twoFabA-like β-hydroxy acyl-ACP dehydrase (DH) domains; (g) at least onechain length factor (CLF) domain; and (h) at least one malonyl-CoA:ACPacyltransferase (MAT) domain. Preferably, the microorganism is anon-bacterial microorganism and more preferably, a eukaryoticmicroorganism.

Yet another embodiment of the present invention relates to amicroorganism comprising a non-bacterial, polyunsaturated fatty acid(PUFA) polyketide synthase (PKS) system, wherein the PKS catalyzes bothiterative and non-iterative enzymatic reactions, and wherein the PUFAPKS system comprises: (a) at least one enoyl ACP-reductase (ER) domain;(b) multiple acyl carrier protein (ACP) domains (at least four); (c) atleast two β-keto acyl-ACP synthase (KS) domains; (d) at least oneacyltransferase (AT) domain; (e) at least one ketoreductase (KR) domain;(f) at least two FabA-like β-hydroxy acyl-ACP dehydrase (DH) domains;(g) at least one chain length factor (CLF) domain; and (h) at least onemalonyl-CoA:ACP acyltransferase (MAT) domain.

In one embodiment of the present invention, it is contemplated that amutagenesis program could be combined with a selective screening processto obtain bioactive molecules of interest. This would include methods tosearch for a range of bioactive compounds. This search would not berestricted to production of those molecules with cis double bonds. Themutagenesis methods could include, but are not limited to: chemicalmutagenesis, gene shuffling, switching regions of the genes encodingspecific enzymatic domains, or mutagenesis restricted to specificregions of those genes, as well as other methods.

For example, high throughput mutagenesis methods could be used toinfluence or optimize production of the desired bioactive molecule. Oncean effective model system has been developed, one could modify thesegenes in a high throughput manner. Utilization of these technologies canbe envisioned on two levels. First, if a sufficiently selective screenfor production of a product of interest (e.g., ARA) can be devised, itcould be used to attempt to alter the system to produce this product(e.g., in lieu of, or in concert with, other strategies such as thosediscussed above). Additionally, if the strategies outlined aboveresulted in a set of genes that did produce the product of interest, thehigh throughput technologies could then be used to optimize the system.For example, if the introduced domain only functioned at relatively lowtemperatures, selection methods could be devised to permit removing thatlimitation. In one embodiment of the invention, screening methods areused to identify additional non-bacterial organisms having novel PKSsystems similar to the PUFA PKS system of Schizochytrium, as describedherein (see above). Homologous PKS systems identified in such organismscan be used in methods similar to those described herein for theSchizochytrium, as well as for an additional source of genetic materialfrom which to create, further modify and/or mutate a PKS system forexpression in that microorganism, in another microorganism, or in ahigher plant, to produce a variety of compounds.

It is recognized that many genetic alterations, either random ordirected, which one may introduce into a native (endogenous, natural)PKS system, will result in an inactivation of enzymatic functions. Apreferred embodiment of the invention includes a system to select foronly those modifications that do not block the ability of the PKS systemto produce a product. For example, the FabB-strain of E. coli isincapable of synthesizing unsaturated fatty acids and requiressupplementation of the medium with fatty acids that can substitute forits normal unsaturated fatty acids in order to grow (see Metz et al.,2001, supra). However, this requirement (for supplementation of themedium) can be removed when the strain is transformed with a functionalPUFA-PKS system (i.e. one that produces a PUFA product in the E. colihost—see (Metz et al., 2001, supra, FIG. 2A). The transformedFabB-strain now requires a functional PUFA-PKS system (to produce theunsaturated fatty acids) for growth without supplementation. The keyelement in this example is that production of a wide range ofunsaturated fatty acid will suffice (even unsaturated fatty acidsubstitutes such as branched chain fatty acids). Therefore, in anotherpreferred embodiment of the invention, one could create a large numberof mutations in one or more of the PUFA PKS genes disclosed herein, andthen transform the appropriately modified FabB-strain (e.g. createmutations in an expression construct containing an ER domain andtransform a FabB-strain having the other essential domains on a separateplasmid—or integrated into the chromosome) and select only for thosetransformants that grow without supplementation of the medium (i.e.,that still possessed an ability to produce a molecule that couldcomplement the FabB-defect). Additional screens could be developed tolook for particular compounds (e.g. use of GC for fatty acids) beingproduced in this selective subset of an active PKS system. One couldenvision a number of similar selective screens for bioactive moleculesof interest.

As described above, in one embodiment of the present invention, agenetically modified microorganism or plant includes a microorganism orplant which has an enhanced ability to synthesize desired bioactivemolecules (products) or which has a newly introduced ability tosynthesize specific products (e.g., to synthesize a specificantibiotic). According to the present invention, “an enhanced ability tosynthesize” a product refers to any enhancement, or up-regulation, in apathway related to the synthesis of the product such that themicroorganism or plant produces an increased amount of the product(including any production of a product where there was none before) ascompared to the wild-type microorganism or plant, cultured or grown,under the same conditions. Methods to produce such genetically modifiedorganisms have been described in detail above.

One embodiment of the present invention is a method to produce desiredbioactive molecules (also referred to as products or compounds) bygrowing or culturing a genetically modified microorganism or plant ofthe present invention (described in detail above). Such a methodincludes the step of culturing in a fermentation medium or growing in asuitable environment, such as soil, a microorganism or plant,respectively, that has a genetic modification as described previouslyherein and in accordance with the present invention. In a preferredembodiment, method to produce bioactive molecules of the presentinvention includes the step of culturing under conditions effective toproduce the bioactive molecule a genetically modified organism thatexpresses a PKS system comprising at least one biologically activedomain of a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS)system. In this preferred aspect, at least one domain of the PUFA PKSsystem is encoded by a nucleic acid sequence selected from the groupconsisting of: (a) a nucleic acid sequence encoding at least one domainof a polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systemfrom a Thraustochytrid microorganism; (b) a nucleic acid sequenceencoding at least one domain of a PUFA PKS system from a microorganismidentified by the novel screening method of the present invention(described above in detail); (c) a nucleic acid sequence encoding anamino acid sequence selected from the group consisting of: SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, and biologically active fragments thereof, (d)a nucleic acid sequence encoding an amino acid sequence selected fromthe group consisting of: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, and biologically active fragmentsthereof, (e) a nucleic acid sequence encoding an amino acid sequencethat is at least about 60% identical to at least 500 consecutive aminoacids of an amino acid sequence selected from the group consisting of:SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6; wherein the amino acidsequence has a biological activity of at least one domain of a PUFA PKSsystem; and, (f) a nucleic acid sequence encoding an amino acid sequencethat is at least about 60% identical to an amino acid sequence selectedfrom the group consisting of: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13,SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26,SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:32; wherein the amino acidsequence has a biological activity of at least one domain of a PUFA PKSsystem. In this preferred aspect of the method, the organism isgenetically modified to affect the activity of the PKS system (describedin detail above). Preferred host cells for genetic modification relatedto the PUFA PKS system of the invention are described above.

In the method of production of desired bioactive compounds of thepresent invention, a genetically modified microorganism is cultured orgrown in a suitable medium, under conditions effective to produce thebioactive compound. An appropriate, or effective, medium refers to anymedium in which a genetically modified microorganism of the presentinvention, when cultured, is capable of producing the desired product.Such a medium is typically an aqueous medium comprising assimilablecarbon, nitrogen and phosphate sources. Such a medium can also includeappropriate salts, minerals, metals and other nutrients. Microorganismsof the present invention can be cultured in conventional fermentationbioreactors. The microorganisms can be cultured by any fermentationprocess which includes, but is not limited to, batch, fed-batch, cellrecycle, and continuous fermentation. Preferred growth conditions forpotential host microorganisms according to the present invention arewell known in the art. The desired bioactive molecules produced by thegenetically modified microorganism can be recovered from thefermentation medium using conventional separation and purificationtechniques. For example, the fermentation medium can be filtered orcentrifuged to remove microorganisms, cell debris and other particulatematter, and the product can be recovered from the cell-free supernatantby conventional methods, such as, for example, ion exchange,chromatography, extraction, solvent extraction, membrane separation,electrodialysis, reverse osmosis, distillation, chemical derivatizationand crystallization. Alternatively, microorganisms producing the desiredcompound, or extracts and various fractions thereof, can be used withoutremoval of the microorganism components from the product.

In the method for production of desired bioactive compounds of thepresent invention, a genetically modified plant is cultured in afermentation medium or grown in a suitable medium such as soil. Anappropriate, or effective, fermentation medium has been discussed indetail above. A suitable growth medium for higher plants includes anygrowth medium for plants, including, but not limited to, soil, sand, anyother particulate media that support root growth (e.g. vermiculite,perlite, etc.) or Hydroponic culture, as well as suitable light, waterand nutritional supplements which optimize the growth of the higherplant. The genetically modified plants of the present invention areengineered to produce significant quantities of the desired productthrough the activity of the PKS system that is genetically modifiedaccording to the present invention. The compounds can be recoveredthrough purification processes which extract the compounds from theplant. In a preferred embodiment, the compound is recovered byharvesting the plant. In this embodiment, the plant can be consumed inits natural state or further processed into consumable products.

As described above, a genetically modified microorganism useful in thepresent invention can, in one aspect, endogenously contain and express aPUFA PKS system, and the genetic modification can be a geneticmodification of one or more of the functional domains of the endogenousPUFA PKS system, whereby the modification has some effect on theactivity of the PUFA PKS system. In another aspect, such an organism canendogenously contain and express a PUFA PKS system, and the geneticmodification can be an introduction of at least one exogenous nucleicacid sequence (e.g., a recombinant nucleic acid molecule), wherein theexogenous nucleic acid sequence encodes at least one biologically activedomain or protein from a second PKS system and/or a protein that affectsthe activity of said PUFA PKS system (e.g., a phosphopantetheinyltransferases (PPTase), discussed below). In yet another aspect, theorganism does not necessarily endogenously (naturally) contain a PUFAPKS system, but is genetically modified to introduce at least onerecombinant nucleic acid molecule encoding an amino acid sequence havingthe biological activity of at least one domain of a PUFA PKS system. Inthis aspect, PUFA PKS activity is affected by introducing or increasingPUFA PKS activity in the organism. Various embodiments associated witheach of these aspects have been discussed in detail above.

In one embodiment of the method to produce bioactive compounds, thegenetic modification changes at least one product produced by theendogenous PKS system, as compared to a wild-type organism.

In another embodiment, the organism endogenously expresses a PKS systemcomprising the at least one biologically active domain of the PUFA PKSsystem, and the genetic modification comprises transfection of theorganism with a recombinant nucleic acid molecule selected from thegroup consisting of: a recombinant nucleic acid molecule encoding atleast one biologically active domain from a second PKS system and arecombinant nucleic acid molecule encoding a protein that affects theactivity of the PUFA PKS system. In this embodiment, the geneticmodification preferably changes at least one product produced by theendogenous PKS system, as compared to a wild-type organism. A second PKSsystem can include another PUFA PKS system (bacterial or non-bacterial),a type I PKS system, a type II PKS system, and/or a modular PKS system.Examples of proteins that affect the activity of a PKS system have beendescribed above (e.g., PPTase).

In another embodiment, the organism is genetically modified bytransfection with a recombinant nucleic acid molecule encoding the atleast one domain of the polyunsaturated fatty acid (PUFA) polyketidesynthase (PKS) system. Such recombinant nucleic acid molecules have beendescribed in detail previously herein.

In another embodiment, the organism endogenously expresses anon-bacterial PUFA PKS system, and the genetic modification comprisessubstitution of a domain from a different PKS system for a nucleic acidsequence encoding at least one domain of the non-bacterial PUFA PKSsystem. In another embodiment, the organism endogenously expresses anon-bacterial PUFA PKS system that has been modified by transfecting theorganism with a recombinant nucleic acid molecule encoding a proteinthat regulates the chain length of fatty acids produced by the PUFA PKSsystem. In one aspect, the recombinant nucleic acid molecule encoding aprotein that regulates the chain length of fatty acids replaces anucleic acid sequence encoding a chain length factor in thenon-bacterial PUFA PKS system. In another aspect, the protein thatregulates the chain length of fatty acids produced by the PUFA PKSsystem is a chain length factor. In another aspect, the protein thatregulates the chain length of fatty acids produced by the PUFA PKSsystem is a chain length factor that directs the synthesis of C20 units.

In another embodiment, the organism expresses a non-bacterial PUFA PKSsystem comprising a genetic modification in a domain selected from thegroup consisting of a domain encoding β-hydroxy acyl-ACP dehydrase (DH)and a domain encoding β-ketoacyl-ACP synthase (KS), wherein themodification alters the ratio of long chain fatty acids produced by thePUFA PKS system as compared to in the absence of the modification. Inone aspect of this embodiment, the modification is selected from thegroup consisting of a deletion of all or a part of the domain, asubstitution of a homologous domain from a different organism for thedomain, and a mutation of the domain.

In another embodiment, the organism expresses a non-bacterial PUFA PKSsystem comprising a modification in an enoyl-ACP reductase (ER) domain,wherein the modification results in the production of a differentcompound as compared to in the absence of the modification. In oneaspect of this embodiment, the modification is selected from the groupconsisting of a deletion of all or a part of the ER domain, asubstitution of an ER domain from a different organism for the ERdomain, and a mutation of the ER domain.

In one embodiment of the method to produce a bioactive molecule, theorganism produces a polyunsaturated fatty acid (PUFA) profile thatdiffers from the naturally occurring organism without a geneticmodification.

Many other genetic modifications useful for producing bioactivemolecules will be apparent to those of skill in the art, given thepresent disclosure, and various other modifications have been discussedpreviously herein. The present invention contemplates any geneticmodification related to a PUFA PKS system as described herein whichresults in the production of a desired bioactive molecule.

Bioactive molecules, according to the present invention, include anymolecules (compounds, products, etc.) that have a biological activity,and that can be produced by a PKS system that comprises at least oneamino acid sequence having a biological activity of at least onefunctional domain of a non-bacterial PUFA PKS system as describedherein. Such bioactive molecules can include, but are not limited to: apolyunsaturated fatty acid (PUFA), an anti-inflammatory formulation, achemotherapeutic agent, an active excipient, an osteoporosis drug, ananti-depressant, an anti-convulsant, an anti-Heliobactor pylori drug, adrug for treatment of neurodegenerative disease, a drug for treatment ofdegenerative liver disease, an antibiotic, and a cholesterol loweringformulation. One advantage of the non-bacterial PUFA PKS system of thepresent invention is the ability of such a system to introducecarbon-carbon double bonds in the cis configuration, and moleculesincluding a double bond at every third carbon. This ability can beutilized to produce a variety of compounds.

Preferably, bioactive compounds of interest are produced by thegenetically modified microorganism in an amount that is greater thanabout 0.05%, and preferably greater than about 0.1%, and more preferablygreater than about 0.25%, and more preferably greater than about 0.5%,and more preferably greater than about 0.75%, and more preferablygreater than about 1%, and more preferably greater than about 2.5%, andmore preferably greater than about 5%, and more preferably greater thanabout 10%, and more preferably greater than about 15%, and even morepreferably greater than about 20% of the dry weight of themicroorganism. For lipid compounds, preferably, such compounds areproduced in an amount that is greater than about 5% of the dry weight ofthe microorganism. For other bioactive compounds, such as antibiotics orcompounds that are synthesized in smaller amounts, those strainspossessing such compounds at of the dry weight of the microorganism areidentified as predictably containing a novel PKS system of the typedescribed above. In some embodiments, particular bioactive molecules(compounds) are secreted by the microorganism, rather than accumulating.Therefore, such bioactive molecules are generally recovered from theculture medium and the concentration of molecule produced will varydepending on the microorganism and the size of the culture.

One embodiment of the present invention relates to a method to modify anendproduct containing at least one fatty acid, comprising adding to saidendproduct an oil produced by a recombinant host cell that expresses atleast one recombinant nucleic acid molecule comprising a nucleic acidsequence encoding at least one biologically active domain of a PUFA PKSsystem. The PUFA PKS system is any non-bacterial PUFA PKS system, andpreferably, is selected from the group of: (a) a nucleic acid sequenceencoding at least one domain of a polyunsaturated fatty acid (PUFA)polyketide synthase (PKS) system from a Thraustochytrid microorganism;(b) a nucleic acid sequence encoding at least one domain of a PUFA PKSsystem from a microorganism identified by the novel screening methoddisclosed herein; (c) a nucleic acid sequence encoding an amino acidsequence selected from the group consisting of: SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, and biologically active fragments thereof, (d) anucleic acid sequence encoding an amino acid sequence selected from thegroup consisting of: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, and biologically active fragmentsthereof; (e) a nucleic acid sequence encoding an amino acid sequencethat is at least about 60% identical to at least 500 consecutive aminoacids of an amino acid sequence selected from the group consisting of:SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6; wherein the amino acidsequence has a biological activity of at least one domain of a PUFA PKSsystem; and, (f) a nucleic acid sequence encoding an amino acid sequencethat is at least about 60% identical to an amino acid sequence selectedfrom the group consisting of: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13,SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26,SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:32; wherein the amino acidsequence has a biological activity of at least one domain of a PUFA PKSsystem. Variations of these nucleic acid sequences have been describedin detail above.

Preferably, the endproduct is selected from the group consisting of afood, a dietary supplement, a pharmaceutical formulation, a humanizedanimal milk, and an infant formula. Suitable pharmaceutical formulationsinclude, but are not limited to, an anti-inflammatory formulation, achemotherapeutic agent, an active excipient, an osteoporosis drug, ananti-depressant, an anti-convulsant, an anti-Heliobactor pylori drug, adrug for treatment of neurodegenerative disease, a drug for treatment ofdegenerative liver disease, an antibiotic, and a cholesterol loweringformulation. In one embodiment, the endproduct is used to treat acondition selected from the group consisting of: chronic inflammation,acute inflammation, gastrointestinal disorder, cancer, cachexia, cardiacrestenosis, neurodegenerative disorder, degenerative disorder of theliver, blood lipid disorder, osteoporosis, osteoarthritis, autoimmunedisease, preeclampsia, preterm birth, age related maculopathy, pulmonarydisorder, and peroxisomal disorder.

Suitable food products include, but are not limited to, fine bakerywares, bread and rolls, breakfast cereals, processed and unprocessedcheese, condiments (ketchup, mayonnaise, etc.), dairy products (milk,yogurt), puddings and gelatine desserts, carbonated drinks, teas,powdered beverage mixes, processed fish products, fruit-based drinks,chewing gum, hard confectionery, frozen dairy products, processed meatproducts, nut and nut-based spreads, pasta, processed poultry products,gravies and sauces, potato chips and other chips or crisps, chocolateand other confectionery, soups and soup mixes, soya based products(milks, drinks, creams, whiteners), vegetable oil-based spreads, andvegetable-based drinks.

Yet another embodiment of the present invention relates to a method toproduce a humanized animal milk. This method includes the steps ofgenetically modifying milk-producing cells of a milk-producing animalwith at least one recombinant nucleic acid molecule comprising a nucleicacid sequence encoding at least one biologically active domain of a PUFAPKS system. The PUFA PKS system is a non-bacterial PUFA PKS system, andpreferably, the at least one domain of the PUFA PKS system is encoded bya nucleic acid sequence selected from the group consisting of: (a) anucleic acid sequence encoding at least one domain of a polyunsaturatedfatty acid (PUFA) polyketide synthase (PKS) system from aThraustochytrid microorganism; (b) a nucleic acid sequence encoding atleast one domain of a PUFA PKS system from a microorganism identified bythe novel screening method described previously herein; (c) a nucleicacid sequence encoding an amino acid sequence selected from the groupconsisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and biologicallyactive fragments thereof, (d) a nucleic acid sequence encoding an aminoacid sequence selected from the group consisting of: SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, andbiologically active fragments thereof; (e) a nucleic acid sequenceencoding an amino acid sequence that is at least about 60% identical toat least 500 consecutive amino acids of an amino acid sequence selectedfrom the group consisting of: SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6;wherein the amino acid sequence has a biological activity of at leastone domain of a PUFA PKS system; and/or (f) a nucleic acid sequenceencoding an amino acid sequence that is at least about 60% identical toan amino acid sequence selected from the group consisting of: SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, and SEQID NO:32; wherein the amino acid sequence has a biological activity ofat least one domain of a PUFA PKS system.

Methods to genetically modify a host cell and to produce a geneticallymodified non-human, milk-producing animal, are known in the art.Examples of host animals to modify include cattle, sheep, pigs, goats,yaks, etc., which are amenable to genetic manipulation and cloning forrapid expansion of a transgene expressing population. For animals,PKS-like transgenes can be adapted for expression in target organelles,tissues and body fluids through modification of the gene regulatoryregions. Of particular interest is the production of PUFAs in the breastmilk of the host animal.

The following examples are provided for the purpose of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Example 1

The following example describes the further analysis of PKS relatedsequences from Schizochytrium.

The present inventors have sequenced the genomic DNA including theentire length of all three open reading frames (Orfs) in theSchizochytrium PUFA PKS system using the general methods outlined inExamples 8 and 9 from PCT Publication No. WO 0042195 and U.S.application Ser. No. 09/231,899. The biologically active domains in theSchizochytrium PKS proteins are depicted graphically in FIG. 1. Thedomain structure of the Schizochytrium PUFA PKS system is described moreparticularly as follows.

Open Reading Frame A (OrfA):

The complete nucleotide sequence for OrfA is represented herein as SEQID NO:1. OrfA is a 8730 nucleotide sequence (not including the stopcodon) which encodes a 2910 amino acid sequence, represented herein asSEQ ID NO:2. Within OrfA are twelve domains:

(a) one β-keto acyl-ACP synthase (KS) domain;

(b) one malonyl-CoA:ACP acyltransferase (MAT) domain;

(c) nine acyl carrier protein (ACP) domains;

(d) one ketoreductase (KR) domain.

The domains contained within OrfA have been determined based on:

(1) results of an analysis with Pfam program (Pfam is a database ofmultiple alignments of protein domains or conserved protein regions. Thealignments represent some evolutionary conserved structure that hasimplications for the protein's function. Profile hidden Markov models(profile HMMs) built from the Pfam alignments can be very useful forautomatically recognizing that a new protein belongs to an existingprotein family, even if the homology is weak. Unlike standard pairwisealignment methods (e.g. BLAST, FASTA), Pfam HMMs deal sensibly withmultidomain proteins. The reference provided for the Pfam version usedis: Bateman A, Birney E, Cerruti L, Durbin R, Etwiller L, Eddy S R,Griffiths-Jones S, Howe K L, Marshall M, Sonnhammer E L (2002) NucleicAcids Research 30(1):276-280); and/or

(2) homology comparison to bacterial PUFA-PKS systems (e.g., Shewanella)using a BLAST 2.0 Basic BLAST homology search using blastp for aminoacid searches with standard default parameters, wherein the querysequence is filtered for low complexity regions by default (described inAltschul, S. F., Madden, T. L., Schääffer, A. A., Zhang, J., Zhang, Z.,Miller, W. & Lipman, D. J. (1997) “Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs.” Nucleic Acids Res.25:3389-3402, incorporated herein by reference in its entirety).

Sequences provided for individual domains are believed to contain thefull length of the sequence encoding a functional domain, and maycontain additional flanking sequence within the Orf.

ORFA-KS

The first domain in OrfA is a KS domain, also referred to herein asORFA-KS. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1 and 40 ofSEQ ID NO:1 (OrfA) to an ending point of between about positions 1428and 1500 of SEQ ID NO:1. The nucleotide sequence containing the sequenceencoding the ORFA-KS domain is represented herein as SEQ ID NO:7(positions 1-1500 of SEQ ID NO:1). The amino acid sequence containingthe KS domain spans from a starting point of between about positions 1and 14 of SEQ ID NO:2 (ORFA) to an ending point of between aboutpositions 476 and 500 of SEQ ID NO:2. The amino acid sequence containingthe ORFA-KS domain is represented herein as SEQ ID NO:8 (positions 1-500of SEQ ID NO:2). It is noted that the ORFA-KS domain contains an activesite motif: DXAC* (*acyl binding site C₂₁₅).

ORFA-MAT

The second domain in OrfA is a MAT domain, also referred to herein asORFA-MAT. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1723 and 1798of SEQ ID NO:1 (OrfA) to an ending point of between about positions 2805and 3000 of SEQ ID NO:1. The nucleotide sequence containing the sequenceencoding the ORFA-MAT domain is represented herein as SEQ ID NO:9(positions 1723-3000 of SEQ ID NO:1). The amino acid sequence containingthe MAT domain spans from a starting point of between about positions575 and 600 of SEQ ID NO:2 (ORFA) to an ending point of between aboutpositions 935 and 1000 of SEQ ID NO:2. The amino acid sequencecontaining the ORFA-MAT domain is represented herein as SEQ ID NO:10(positions 575-1000 of SEQ ID NO:2). It is noted that the ORFA-MATdomain contains an active site motif: GHS*XG (*acyl binding site S₇₀₆),represented herein as SEQ ID NO:11.

ORFA-ACP#1-9

Domains 3-11 of OrfA are nine tandem ACP domains, also referred toherein as ORFA-ACP (the first domain in the sequence is ORFA-ACP 1, thesecond domain is ORFA-ACP2, the third domain is ORFA-ACP3, etc.). Thefirst ACP domain, ORFA-ACP1, is contained within the nucleotide sequencespanning from about position 3343 to about position 3600 of SEQ ID NO:1(OrfA). The nucleotide sequence containing the sequence encoding theORFA-ACP1 domain is represented herein as SEQ ID NO:12 (positions3343-3600 of SEQ ID NO:1). The amino acid sequence containing the firstACP domain spans from about position 1115 to about position 1200 of SEQID NO:2. The amino acid sequence containing the ORFA-ACP 1 domain isrepresented herein as SEQ ID NO:13 (positions 1115-1200 of SEQ ID NO:2).It is noted that the ORFA-ACP1 domain contains an active site motif:LGIDS* (*pantetheine binding motif S₁₁₅₇), represented herein by SEQ IDNO:14. The nucleotide and amino acid sequences of all nine ACP domainsare highly conserved and therefore, the sequence for each domain is notrepresented herein by an individual sequence identifier. However, basedon this information, one of skill in the art can readily determine thesequence for each of the other eight ACP domains. The repeat intervalfor the nine domains is approximately about 110 to about 330 nucleotidesof SEQ ID NO:1.

All nine ACP domains together span a region of OrfA of from aboutposition 3283 to about position 6288 of SEQ ID NO:1, which correspondsto amino acid positions of from about 1095 to about 2096 of SEQ ID NO:2.This region includes the linker segments between individual ACP domains.Each of the nine ACP domains contains a pantetheine binding motif LGIDS*(represented herein by SEQ ID NO:14), wherein * is the pantetheinebinding site S. At each end of the ACP domain region and between eachACP domain is a region that is highly enriched for proline (P) andalanine (A), which is believed to be a linker region. For example,between ACP domains 1 and 2 is the sequence: APAPVKAAAPAAPVASAPAPA,represented herein as SEQ ID NO:15.

ORFA-KR

Domain 12 in OrfA is a KR domain, also referred to herein as ORFA-KR.This domain is contained within the nucleotide sequence spanning from astarting point of about position 6598 of SEQ ID NO:1 to an ending pointof about position 8730 of SEQ ID NO:1. The nucleotide sequencecontaining the sequence encoding the ORFA-KR domain is representedherein as SEQ ID NO:17 (positions 6598-8730 of SEQ ID NO:1). The aminoacid sequence containing the KR domain spans from a starting point ofabout position 2200 of SEQ ID NO:2 (ORFA) to an ending point of aboutposition 2910 of SEQ ID NO:2. The amino acid sequence containing theORFA-KR domain is represented herein as SEQ ID NO:18 (positions2200-2910 of SEQ ID NO:2). Within the KR domain is a core region withhomology to short chain aldehyde-dehydrogenases (KR is a member of thisfamily). This core region spans from about position 7198 to aboutposition 7500 of SEQ ID NO:1, which corresponds to amino acid positions2400-2500 of SEQ ID NO:2.

Open Reading Frame B (OrfB):

The complete nucleotide sequence for OrfB is represented herein as SEQID NO:3. OrfB is a 6177 nucleotide sequence (not including the stopcodon) which encodes a 2059 amino acid sequence, represented herein asSEQ ID NO:4. Within OrfB are four domains:

-   -   (a) β-keto acyl-ACP synthase (KS) domain;    -   (b) one chain length factor (CLF) domain;    -   (c) one acyl transferase (AT) domain;    -   (d) one enoyl ACP-reductase (ER) domain.

The domains contained within ORFB have been determined based on: (1)results of an analysis with Pfam program, described above; and/or (2)homology comparison to bacterial PUFA-PKS systems (e.g., Shewanella)using a BLAST 2.0 Basic BLAST homology search, also described above.Sequences provided for individual domains are believed to contain thefull length of the sequence encoding a functional domain, and maycontain additional flanking sequence within the Orf.

ORFB-KS

The first domain in OrfB is a KS domain, also referred to herein asORFB-KS. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1 and 43 ofSEQ ID NO:3 (OrfB) to an ending point of between about positions 1332and 1350 of SEQ ID NO:3. The nucleotide sequence containing the sequenceencoding the ORFB-KS domain is represented herein as SEQ ID NO:19(positions 1-1350 of SEQ ID NO:3). The amino acid sequence containingthe KS domain spans from a starting point of between about positions 1and 15 of SEQ ID NO:4 (ORFB) to an ending point of between aboutpositions 444 and 450 of SEQ ID NO:4. The amino acid sequence containingthe ORFB-KS domain is represented herein as SEQ ID NO:20 (positions1-450 of SEQ ID NO:4). It is noted that the ORFB-KS domain contains anactive site motif: DXAC* (*acyl binding site C₁₉₆).

ORFB-CLF

The second domain in OrfB is a CLF domain, also referred to herein asORFB-CLF. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1378 and 1402of SEQ ID NO:3 (OrfB) to an ending point of between about positions 2682and 2700 of SEQ ID NO:3. The nucleotide sequence containing the sequenceencoding the ORFB-CLF domain is represented herein as SEQ ID NO:21(positions 1378-2700 of SEQ ID NO:3). The amino acid sequence containingthe CLF domain spans from a starting point of between about positions460 and 468 of SEQ ID NO:4 (ORFB) to an ending point of between aboutpositions 894 and 900 of SEQ ID NO:4. The amino acid sequence containingthe ORFB-CLF domain is represented herein as SEQ ID NO:22 (positions460-900 of SEQ ID NO:4). It is noted that the ORFB-CLF domain contains aKS active site motif without the acyl-binding cysteine.

ORFB-AT

The third domain in OrfB is an AT domain, also referred to herein asORFB-AT. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 2701 and 3598of SEQ ID NO:3 (OrfB) to an ending point of between about positions 3975and 4200 of SEQ ID NO:3. The nucleotide sequence containing the sequenceencoding the ORFB-AT domain is represented herein as SEQ ID NO:23(positions 2701-4200 of SEQ ID NO:3). The amino acid sequence containingthe AT domain spans from a starting point of between about positions 901and 1200 of SEQ ID NO:4 (ORFB) to an ending point of between aboutpositions 1325 and 1400 of SEQ ID NO:4. The amino acid sequencecontaining the ORFB-AT domain is represented herein as SEQ ID NO:24(positions 901-1400 of SEQ ID NO:4). It is noted that the ORFB-AT domaincontains an AT active site motif of GxS*xG (*acyl binding site S₁₁₄₀).

ORFB-ER

The fourth domain in OrfB is an ER domain, also referred to herein asORFB-ER. This domain is contained within the nucleotide sequencespanning from a starting point of about position 4648 of SEQ ID NO:3(OrfB) to an ending point of about position 6177 of SEQ ID NO:3. Thenucleotide sequence containing the sequence encoding the ORFB-ER domainis represented herein as SEQ ID NO:25 (positions 4648-6177 of SEQ IDNO:3). The amino acid sequence containing the ER domain spans from astarting point of about position 1550 of SEQ ID NO:4 (ORFB) to an endingpoint of about position 2059 of SEQ ID NO:4. The amino acid sequencecontaining the ORFB-ER domain is represented herein as SEQ ID NO:26(positions 1550-2059 of SEQ ID NO:4).

Open Reading Frame C (OrfC):

The complete nucleotide sequence for OrfC is represented herein as SEQID NO:5. OrfC is a 4509 nucleotide sequence (not including the stopcodon) which encodes a 1503 amino acid sequence, represented herein asSEQ ID NO:6. Within OrfC are three domains:

-   -   (a) two FabA-like β-hydroxy acyl-ACP dehydrase (DH) domains;    -   (b) one enoyl ACP-reductase (ER) domain.

The domains contained within ORFC have been determined based on: (1)results of an analysis with Pfam program, described above; and/or (2)homology comparison to bacterial PUFA-PKS systems (e.g., Shewanella)using a BLAST 2.0 Basic BLAST homology search, also described above.Sequences provided for individual domains are believed to contain thefull length of the sequence encoding a functional domain, and maycontain additional flanking sequence within the Orf.

ORFC-DH1

The first domain in OrfC is a DH domain, also referred to herein asORFC-DH1. This is one of two DH domains in OrfC, and therefore isdesignated DH1. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1 and 778 ofSEQ ID NO:5 (OrfC) to an ending point of between about positions 1233and 1350 of SEQ ID NO:5. The nucleotide sequence containing the sequenceencoding the ORFC-DH1 domain is represented herein as SEQ ID NO:27(positions 1-1350 of SEQ ID NO:5). The amino acid sequence containingthe DH1 domain spans from a starting point of between about positions 1and 260 of SEQ ID NO:6 (ORFC) to an ending point of between aboutpositions 411 and 450 of SEQ ID NO:6. The amino acid sequence containingthe ORFC-DH1 domain is represented herein as SEQ ID NO:28 (positions1-450 of SEQ ID NO:6).

ORFC-DH2

The second domain in OrfC is a DH domain, also referred to herein asORFC-DH2. This is the second of two DH domains in OrfC, and therefore isdesignated DH2. This domain is contained within the nucleotide sequencespanning from a starting point of between about positions 1351 and 2437of SEQ ID NO:5 (OrfC) to an ending point of between about positions 2607and 2850 of SEQ ID NO:5. The nucleotide sequence containing the sequenceencoding the ORFC-DH2 domain is represented herein as SEQ ID NO:29(positions 1351-2850 of SEQ ID NO:5). The amino acid sequence containingthe DH2 domain spans from a starting point of between about positions451 and 813 of SEQ ID NO:6 (ORFC) to an ending point of between aboutpositions 869 and 950 of SEQ ID NO:6. The amino acid sequence containingthe ORFC-DH2 domain is represented herein as SEQ ID NO:30 (positions451-950 of SEQ ID NO:6).

ORFC-ER

The third domain in OrfC is an ER domain, also referred to herein asORFC-ER. This domain is contained within the nucleotide sequencespanning from a starting point of about position 2998 of SEQ ID NO:5(OrfC) to an ending point of about position 4509 of SEQ ID NO:5. Thenucleotide sequence containing the sequence encoding the ORFC-ER domainis represented herein as SEQ ID NO:31 (positions 2998-4509 of SEQ IDNO:5). The amino acid sequence containing the ER domain spans from astarting point of about position 1000 of SEQ ID NO:6 (ORFC) to an endingpoint of about position 1502 of SEQ ID NO:6. The amino acid sequencecontaining the ORFC-ER domain is represented herein as SEQ ID NO:32(positions 1000-1502 of SEQ ID NO:6).

Example 2

The following example describes the use of the screening process of thepresent invention to identify three other non-bacterial organismscomprising a PUFA PKS system according to the present invention.

Thraustochytrium sp. 23B (ATCC 20892) was cultured according to thescreening method described in U.S. Provisional Application Ser. No.60/298,796 and as described in detail herein.

The biorational screen (using shake flask cultures) developed fordetecting microorganisms containing PUFA producing PKS systems is asfollows:

Two mL of a culture of the strain/microorganism to be tested is placedin 250 mL baffled shake flask with 50 mL culture media (aerobictreatment) and another 2 mL of culture of the same strain is placed in a250 mL non-baffled shake flask with 200 mL culture medium (anoxictreatment). Both flasks are placed on a shaker table at 200 rpm. After48-72 hr of culture time, the cultures are harvested by centrifugationand the cells analyzed for fatty acid methyl esters via gaschromatography to determine the following data for each culture: (1)fatty acid profile; (2) PUFA content; (3) fat content (estimated asamount total fatty acids (TFA)).

These data are then analyzed asking the following five questions:

Selection Criteria Low O₂/Anoxic Flask vs. Aerobic Flask (Yes/No)

(1) Did the DHA (or other PUFA content) (as % FAME) stay about the sameor preferably increase in the low oxygen culture compared to the aerobicculture?

(2) Is C14:0+C16:0+C16:1 greater than about 40% TFA in the anoxicculture?

(3) Is there very little (>1% as FAME) or no precursors(C18:3n-3+C18:2n-6+C18:3n-6) to the conventional oxygen dependentelongase/desaturase pathway in the anoxic culture?

(4) Did fat content (as amount total fatty acids/cell dry weight)increase in the low oxygen culture compared to the aerobic culture?

(5) Did DHA (or other PUFA content) increase as % cell dry weight in thelow oxygen culture compared to the aerobic culture?

If first three questions are answered yes, there is a good indicationthat the strain contains a PKS genetic system for making long chainPUFAs. The more questions that are answered yes (preferably the firstthree questions must be answered yes), the stronger the indication thatthe strain contains such a PKS genetic system. If all five questions areanswered yes, then there is a very strong indication that the straincontains a PKS genetic system for making long chain PUFAs.

Following the method outlined above, a frozen vial of Thraustochytriumsp. 23B (ATCC 20892) was used to inoculate a 250 mL shake flaskcontaining 50 mL of RCA medium. The culture was shaken on a shaker table(200 rpm) for 72 hr at 25° C. RCA medium contains the following:

RCA Medium Deionized water 1000 mL Reef Crystals ® sea salts 40 g/LGlucose 20 g/L Monosodium glutamate (MSG) 20 g/L Yeast extract 1 g/L PIImetals* 5 mL/L Vitamin mix* 1 mL/L pH 7.0 *PII metal mix and vitamin mixare same as those outlined in U.S. Pat. No. 5,130,742, incorporatedherein by reference in its entirety.

25 mL of the 72 hr old culture was then used to inoculate another 250 mLshake flask containing 50 mL of low nitrogen RCA medium (10 g/L MSGinstead of 20 g/L) and the other 25 mL of culture was used to inoculatea 250 mL shake flask containing 175 mL of low-nitrogen RCA medium. Thetwo flasks were then placed on a shaker table (200 rpm) for 72 hr at 25°C. The cells were then harvested via centrifugation and dried bylyophilization. The dried cells were analyzed for fat content and fattyacid profile and content using standard gas chromatograph procedures(such as those outlined in U.S. Pat. No. 5,130,742).

The screening results for Thraustochytrium 23B were as follows:

Did DHA as % FAME increase? Yes (38−>44%) C14:0 + C16:0 + C16:1 greaterthan about Yes (44%) 40% TFA? No C18:3(n-3) or C18:3(n-6)? Yes (0%) Didfat content increase? Yes (2-fold increase) Did DHA (or other HUFAcontent increase)? Yes (2.3-fold increase)

The results, especially the significant increase in DHA content (as %FAME) under low oxygen conditions, conditions, strongly indicates thepresence of a PUFA producing PKS system in this strain ofThraustochytrium.

In order to provide additional data confirming the presence of a PUFAPKS system, southern blot of Thraustochytrium 23B was conducted usingPKS probes from Schizochytrium strain 20888, a strain which has alreadybeen determined to contain a PUFA producing PKS system (i.e., SEQ IDNos:1-32 described above). Fragments of Thraustochytrium 23B genomic DNAwhich are homologous to hybridization probes from PKS PUFA synthesisgenes were detected using the Southern blot technique. Thraustochytrium23B genomic DNA was digested with either ClaI or KpnI restrictionendonucleases, separated by agarose gel electrophoresis (0.7% agarose,in standard Tris-Acetate-EDTA buffer), and blotted to a Schleicher &Schuell Nytran Supercharge membrane by capillary transfer. Twodigoxigenin labeled hybridization probes were used—one specific for theEnoyl Reductase (ER) region of Schizochytrium PKS Orf B (nucleotides5012-5511 of Orf B; SEQ ID NO:3), and the other specific for a conservedregion at the beginning of Schizochytrium PKS Orf C (nucleotides 76-549of OrfC; SEQ ID NO:5).

The OrfB-ER probe detected an approximately 13 kb ClaI fragment and anapproximately 3.6 kb KpnI fragment in the Thraustochytrium 23B genomicDNA. The OrfC probe detected an approximately 7.5 kb ClaI fragment andan approximately 4.6 kb KpnI fragment in the Thraustochytrium 23Bgenomic DNA.

Finally, a recombinant genomic library, consisting of DNA fragments fromThraustochytrium 23B genomic DNA inserted into vector lambda FIX II(Stratagene), was screened using digoxigenin labeled probescorresponding to the following segments of Schizochytrium 20888 PUFA-PKSgenes: nucleotides 7385-7879 of Orf A (SEQ ID NO:1), nucleotides5012-5511 of Orf B (SEQ ID NO:3), and nucleotides 76-549 of Orf C (SEQID NO:5). Each of these probes detected positive plaques from theThraustochytrium 23B library, indicating extensive homology between theSchizochytrium PUFA-PKS genes and the genes of Thraustochytrium 23B.

In summary, these results demonstrate that Thraustochytrium 23B genomicDNA contains sequences that are homologous to PKS genes fromSchizochytrium 20888.

This Thraustochytrid microorganism is encompassed herein as anadditional sources of these genes for use in the embodiments above.

Thraustochytrium 23B (ATCC 20892) is significantly different fromSchizochytrium sp. (ATCC 20888) in its fatty acid profile.Thraustochytrium 23B can have DHA:DPA(n-6) ratios as high as 14:1compared to only 2-3:1 in Schizochytrium (ATCC 20888). Thraustochytrium23B can also have higher levels of C20:5(n-3). Analysis of the domainsin the PUFA PKS system of Thraustochytrium 23B in comparison to theknown Schizochytrium PUFA PKS system should provide us with keyinformation on how to modify these domains to influence the ratio andtypes of PUFA produced using these systems.

The screening method described above has been utilized the identifyother potential candidate strains containing a PUFA PKS system. Twoadditional strains that have been identified by the present inventors tohave PUFA PKS systems are Schizochytrium limacium (SR21) Honda & Yokochi(IFO32693) and Ulkenia (BP-5601). Both were screened as above but in N2media (glucose: 60 g/L; KH₂PO₄: 4.0 g/l; yeast extract: 1.0 g/L; cornsteep liquor: 1 mL/L; NH₄NO₃: 1.0 g/L; artificial sea salts (ReefCrystals): 20 g/L; all above concentrations mixed in deionized water).For both the Schizochytrium and Ulkenia strains, the answers to thefirst three screen questions discussed above for Thraustochytrium 23Bwas yes (Schizochytrium—DHA % FAME 32->41% aerobic vs anoxic, 58%14:0/16:0/16:1, 0% precursors) and (Ulkenia—DHA % FAME 28->44% aerobicvs anoxic, 63% 14:0/16:0/16:1, 0% precursors), indicating that thesestrains are good candidates for containing a PUFA PKS system. Negativeanswers were obtained for the final two questions for each strain: fatdecreased from 61% dry wt to 22% dry weight, and DHA from 21-9% dryweight in S. limacium and fat decreased from 59 to 21% dry weight inUlkenia and DHA from 16% to 9% dry weight. These Thraustochytridmicroorganisms are also claimed herein as additional sources of thegenes for use in the embodiments above.

Example 3

The following example demonstrates that DHA and DPA synthesis inSchizochytrium does not involve membrane-bound desaturases or fatty acidelongation enzymes like those described for other eukaryotes(Parker-Barnes et al., 2000, supra; Shanklin et al., 1998, supra).

Schizochytrium accumulates large quantities of triacylglycerols rich inDHA and docosapentaenoic acid (DPA; 22:5ω6); e.g., 30% DHA+DPA by dryweight. In eukaryotes that synthesize 20- and 22-carbon PUFAs by anelongation/desaturation pathway, the pools of 18-, 20- and 22-carbonintermediates are relatively large so that in vivo labeling experimentsusing [¹⁴C]-acetate reveal clear precursor-product kinetics for thepredicted intermediates. Furthermore, radiolabeled intermediatesprovided exogenously to such organisms are converted to the final PUFAproducts.

[1-¹⁴C]acetate was supplied to a 2-day-old culture as a single pulse atzero time. Samples of cells were then harvested by centrifugation andthe lipids were extracted. In addition, [1-¹⁴C]acetate uptake by thecells was estimated by measuring the radioactivity of the sample beforeand after centrifugation. Fatty acid methyl esters derived from thetotal cell lipids were separated by AgNO₃-TLC (solvent, hexane:diethylether:acetic acid, 70:30:2 by volume). The identity of the fatty acidbands was verified by gas chromatography, and the radioactivity in themwas measured by scintillation counting. Results showed that[1-¹⁴C]-acetate was rapidly taken up by Schizochytrium cells andincorporated into fatty acids, but at the shortest labeling time (1 min)DHA contained 31% of the label recovered in fatty acids and thispercentage remained essentially unchanged during the 10-15 min of[¹⁴C]-acetate incorporation and the subsequent 24 hours of culturegrowth (data not shown). Similarly, DPA represented 10% of the labelthroughout the experiment. There is no evidence for a precursor-productrelationship between 16- or 18-carbon fatty acids and the 22-carbonpolyunsaturated fatty acids. These results are consistent with rapidsynthesis of DHA from [¹⁴C]-acetate involving very small (possiblyenzyme-bound) pools of intermediates.

Next, cells were disrupted in 100 mM phosphate buffer (pH 7.2),containing 2 mM DTT, 2 mM EDTA, and 10% glycerol, by vortexing withglass beads. The cell-free homogenate was centrifuged at 100,000 g for 1hour. Equivalent aliquots of total homogenate, pellet (H-S pellet), andsupernatant (H-S super) fractions were incubated in homogenizationbuffer supplemented with 20 μM acetyl-CoA, 100 μM [1-¹⁴C]malonyl-CoA(0.9 Gbq/mol), 2 mM NADH, and 2 mM NADPH for 60 min at 25° C. Assayswere extracted and fatty acid methyl esters were prepared and separatedas described above before detection of radioactivity with anInstantimager (Packard Instruments, Meriden, Conn.). Results showed thata cell-free homogenate derived from Schizochytrium cultures incorporated[1-¹⁴C]-malonyl-CoA into DHA, DPA, and saturated fatty acids (data notshown). The same biosynthetic activities were retained by a 10,000×gsupernatant fraction but were not present in the membrane pellet. Thesedata contrast with those obtained during assays of the bacterial enzymes(see Metz et al., 2001, supra) and may indicate use of a different(soluble) acyl acceptor molecule. Thus, DHA and DPA synthesis inSchizochytrium does not involve membrane-bound desaturases or fatty acidelongation enzymes like those described for other eukaryotes.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1. An isolated nucleic acid molecule comprising a nucleic acid sequenceencoding an amino acid sequence that is at least 95% identical to SEQ IDNO:4 or that is an enzymatically active fragment of SEQ ID NO:4, whereinsaid amino acid sequence has β-keto acyl-ACP synthase (KS) activity,chain length factor (CLF) activity, acyl transferase (AT) activity, andenoyl ACP-reductase (ER) activity.
 2. The isolated nucleic acid moleculeof claim 1, comprising a nucleic acid sequence encoding an amino acidsequence that is an enzymatically active fragment of SEQ ID NO:4,wherein said amino acid sequence has β-keto acyl-ACP synthase (KS)activity, chain length factor (CLF) activity, acyl transferase (AT)activity, and enoyl ACP-reductase (ER) activity.
 3. The isolated nucleicacid molecule of claim 1, comprising a nucleic acid sequence encoding anamino acid sequence that is at least 95% identical to SEQ ID NO:4,wherein said amino acid sequence has β-keto acyl-ACP synthase (KS)activity, chain length factor (CLF) activity, acyl transferase (AT)activity, and enoyl ACP-reductase (ER) activity.
 4. The isolated nucleicacid molecule of claim 1, consisting of a nucleic acid sequence encodingan amino acid sequence that is at least 95% identical to SEQ ID NO:4,wherein said amino acid sequence has β-keto acyl-ACP synthase (KS)activity, chain length factor (CLF) activity, acyl transferase (AT)activity, and enoyl ACP-reductase (ER) activity.
 5. The isolated nucleicacid molecule of claim 1, comprising a nucleic acid sequence encoding anamino acid sequence that is at least 96% identical to SEQ ID NO:4wherein said amino acid sequence has β-keto acyl-ACP synthase (KS)activity, chain length factor (CLF) activity, acyl transferase (AT)activity, and enoyl ACP-reductase (ER) activity.
 6. The isolated nucleicacid molecule of claim 1, consisting of a nucleic acid sequence encodingan amino acid sequence that is at least 96% identical to SEQ ID NO:4wherein said amino acid sequence has β-keto acyl-ACP synthase (KS)activity, chain length factor (CLF) activity, acyl transferase (AT)activity, and enoyl ACP-reductase (ER) activity.
 7. The isolated nucleicacid molecule of claim 1, comprising a nucleic acid sequence encoding anamino acid sequence that is at least 97% identical to SEQ ID NO:4wherein said amino acid sequence has β-keto acyl-ACP synthase (KS)activity, chain length factor (CLF) activity, acyl transferase (AT)activity, and enoyl ACP-reductase (ER) activity.
 8. The isolated nucleicacid molecule of claim 1, consisting of a nucleic acid sequence encodingan amino acid sequence that is at least 97% identical to SEQ ID NO:4wherein said amino acid sequence has β-keto acyl-ACP synthase (KS)activity, chain length factor (CLF) activity, acyl transferase (AT)activity, and enoyl ACP-reductase (ER) activity.
 9. The isolated nucleicacid molecule of claim 1, comprising a nucleic acid sequence encoding anamino acid sequence that is at least 98% identical to SEQ ID NO:4wherein said amino acid sequence has β-keto acyl-ACP synthase (KS)activity, chain length factor (CLF) activity, acyl transferase (AT)activity, and enoyl ACP-reductase (ER) activity.
 10. The isolatednucleic acid molecule of claim 1, consisting of a nucleic acid sequenceencoding an amino acid sequence that is at least 98% identical to SEQ IDNO:4 wherein said amino acid sequence has β-keto acyl-ACP synthase (KS)activity, chain length factor (CLF) activity, acyl transferase (AT)activity, and enoyl ACP-reductase (ER) activity.
 11. The isolatednucleic acid molecule of claim 1, comprising a nucleic acid sequenceencoding an amino acid sequence that is at least 99% identical to SEQ IDNO:4, wherein said amino acid sequence has β-keto acyl-ACP synthase (KS)activity, chain length factor (CLF) activity, acyl transferase (AT)activity, and enoyl ACP-reductase (ER) activity.
 12. The isolatednucleic acid molecule of claim 1, consisting of a nucleic acid sequenceencoding an amino acid sequence that is at least 99% identical to SEQ IDNO:4, wherein said amino acid sequence has β-keto acyl-ACP synthase (KS)activity, chain length factor (CLF) activity, acyl transferase (AT)activity, and enoyl ACP-reductase (ER) activity.
 13. The isolatednucleic acid molecule of claim 1, comprising a nucleic acid sequenceencoding the amino acid sequence of SEQ ID NO:4.
 14. The isolatednucleic acid molecule of claim 1, consisting of a nucleic acid sequenceencoding the amino acid sequence of SEQ ID NO:4.
 15. The isolatednucleic acid molecule of claim 1, wherein the nucleic acid moleculecomprises SEQ ID NO:3.
 16. The isolated nucleic acid molecule of claim1, wherein the nucleic acid molecule consists of SEQ ID NO:3.
 17. Arecombinant nucleic acid molecule comprising the nucleic acid moleculeof claim 1 and a transcription control sequence.
 18. A recombinant plantcell that expresses the nucleic acid molecule of claim
 1. 19. Arecombinant microbial cell that expresses a recombinant vectorcomprising the nucleic acid molecule of claim 1 and a transcriptioncontrol sequence.
 20. The recombinant microbial cell of claim 19,wherein the microbial cell is a bacterium.
 21. The recombinant microbialcell of claim 19, wherein the microbial cell is a Thraustochytrialesmicroorganism.
 22. The recombinant microbial cell of claim 21, whereinthe Thraustochytriales microorganism is a Schizochytrium or aThraustochytrium.
 23. A method to produce at least one polyunsaturatedfatty acid (PUFA), comprising culturing under conditions effective toproduce the PUFA, a plant cell or a microbial cell that expresses a PKSsystem for production of PUFAs, wherein the plant cell or microbial cellexpresses a recombinant vector comprising the nucleic acid molecule ofclaim 1.